Epstein-barr virus antibodies and uses thereof

ABSTRACT

Disclosed herein are antibodies or immunogenic fragments thereof that specifically bind to Epstein-Barr virus (EBV) glycoprotein 350 (gp350) or 220 or one or more immunogenic peptides. Also disclosed are immunogenic peptides comprising fragments of gp350 amino acid sequence, EBV antibody-small molecule conjugates and pharmaceutical compositions comprising the antibody or an immunogenic fragment thereof, one or more immunogenic peptides, or the EBV antibody-small molecule conjugate. The antibodies, immunogenic peptides, conjugates, and pharmaceutical compositions can be used to treat or prevent EBV infections and EBV-associated conditions and diseases.

PRIORITY CLAIM

The present invention is a continuation of U.S. patent application Ser.No. 16/940,304, filed on Jul. 27, 2020, which is a continuation-in-partof U.S. patent application Ser. No. 16/609,078, filed on Oct. 28, 2019,which is a national phase entry of International Application No.PCT/US2018/30030, filed on Apr. 27, 2018, which claims priority to U.S.Provisional Patent Application No. 62/491,945, filed on Apr. 28, 2017.U.S. patent application Ser. No. 16/940,304 also claims the benefit ofU.S. Provisional Patent Application No. 62/880,024, filed on Jul. 29,2019. The contents of all priority applications are incorporated hereinby reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with government support under Grant Nos.R21CA205106 and R21CA232275, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

SEQUENCE LISTING

This disclosure includes a sequence listing, which is submitted in ASCIIformat via EFS-Web, and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jun. 16, 2022, is namedSequenceListing.txt and is 83 kilobytes in size.

BACKGROUND

Epstein-Barr virus (EBV) infection is the causal agent of acuteinfectious mononucleosis (62, 63). Persistent EBV infection inimmunodeficient individuals is associated with numerous epithelial andlymphoid malignancies, such as nasopharyngeal carcinoma, gastriccarcinoma, Burkitt lymphoma, Hodgkin lymphoma, and post-transplantlymphoproliferative diseases (PTLD) (1). Transplantation is thetreatment of choice for a variety of patients with end-stage organfailure or hematologic malignancies, or in need of reconstructivetransplantation (1). Transplantation success depends entirely on potentimmunosuppressive drugs to prevent stem cell/organ rejection. However,these drugs impose several serious side effects, including an increasedrisk of infection with or reactivation of Epstein-Barr virus (EBV), andthe resultant development of PTLDs, which are aggressive,life-threatening complications (2, 3). Through the early 2000s, PTLDpatients who had been EBV-naïve prior to transplantation showedmortality rates of 50-90% for stem cell and solid organ transplants;while recent data suggest outcomes have improved, challenges remain.PTLDs usually develop in EBV-naïve patients, particularly pediatricpatients, who receive organs from EBV+ donors. A variety ofnon-standardized, non-specific treatments are used to treat EBV+ PTLDcases (4-9). Initial clinical management typically involves reduction ofimmunosuppression; however, this can lead to graft-versus-host disease.Other treatments including radiation/chemotherapy and excision of PTLDlesions all have undesirable side effects. Second-line treatment oftenincludes antibodies (Abs) against the B cell antigen, CD20; however,this also targets healthy B cells, further weakening the immune systemand exposing patients to other opportunistic infections.

In over 50 years of EBV vaccine research, few candidates havedemonstrated partial clinical efficacy, and none have been efficaciousenough to elicit sterilizing immunity and be licensed (24). Antibodies,whether elicited in the host naturally or via passive immunization,provide an effective first-line of defense against viral infection.

Thus, there is an urgent need for a novel EBV-specific therapy thattargets EBV+ cells to neutralize EBV infection and prevent subsequentPTLD development in EBV-naïve patients.

SUMMARY

In one aspect, this disclosure relates to an Epstein-Barr virus (EBV)antibody or an immunogenic fragment thereof. In some embodiments, theEBV antibody or an immunogenic fragment thereof specifically binds toEBV glycoprotein 350/220. In some embodiments, the EBV antibodycomprises a VH region comprising CDR-1, CDR-2, and CDR-3 represented bySEQ ID NOs: 5-19, 21-35, and 37-51, respectively. In some embodiments,the EBV antibody comprises a VL region comprising CDR-1, CDR-2, andCDR-3 represented by SEQ ID NOs: 53-67, 69-83, and 85-99, respectively.In some embodiments, the EBV antibody is a monoclonal antibody. In someembodiments, the EBV antibody is a chimeric antibody, a human antibody,or a humanized antibody. In some embodiments, the EBV antibody is aneutralizing antibody. In some embodiments, the EBV antibody ishumanized 72A1 or humanized E1D1. For example, the EBV antibodycomprises one or more CDRs of antibody clone 72A1. In another example,the EBV antibody comprises one or more CDRs of antibody clone E1D1. Insome embodiments, the EBV antibody comprises a heavy chain having anamino acid sequence of SEQ ID NO: 180, SEQ ID NO: 185, or a sequence atleast 90%, at least 95%, at least 98%, or at least 99% identical to SEQID NO: 180 or SEQ ID NO: 185. In some embodiments, the EBV antibodycomprises a light chain having an amino acid sequence of SEQ ID NO: 181,SEQ ID NO: 186, or a sequence at least 90%, at least 95%, at least 98%,or at least 99% identical to SEQ ID NO: 181 or SEQ ID NO: 186.

In another aspect, this disclosure relates to an immunogenic peptidecomprising the amino acid sequence of a fragment of EBV350 such asAA1-101, AA102-201, or AA402-501, or an amino acid sequence identical toor sharing at least 60% similarity to the fragment. In some embodiments,the immunogenic peptide further comprises a known immunogenic peptidesuch as keyhole limpet hemocyanin (KLH) peptide.

In another aspect, this disclosure relates to an EBV antibody-smallmolecule conjugate. The EBV antibodies disclosed herein can beconjugated to small molecules having activities against EBV-transformedcells. For example, the small molecules have anti-proliferativeactivities against EBV-transformed B lymphoma cells. In someembodiments, the small molecules are growth inhibitors of EBV infected Bcells. In some embodiments, the small molecule is L₂P₄, 2-butynediamide,or a derivative thereof. In some embodiments, the small molecule isconjugated to the antibody via a linker or an adaptor. In someembodiments, the small molecule is conjugated to the constant region ofthe heavy chain or the light chain of the antibody.

In a related aspect, this disclosure relates to a pharmaceuticalcomposition comprising one or more EBV antibodies disclosed herein orone or more immunogenic fragments thereof, one or more immunogenicpeptides disclosed herein, or the EBV antibody-small molecule conjugatedisclosed herein. The pharmaceutical composition can further compriseone or more pharmaceutically acceptable excipients. The pharmaceuticalcomposition can be formulated into any suitable formulation depending onthe administration route. In some embodiments, the pharmaceuticalcomposition comprising a humanized 72A1, a humanized E1D1, or both.

In another aspect, this disclosure relates to a method of neutralizingEBV infection. The method includes administering to a subject infectedwith EBV a therapeutically effective amount of one or more EBVantibodies disclosed herein or one or more immunogenic fragmentsthereof, the EBV antibody-small molecule conjugate, one or moreimmunogenic peptides disclosed herein, or the pharmaceutical compositiondescribed above. In some embodiments, the subject is human. In someembodiments, the subject suffers from or at an elevated risk ofsuffering from EBV infection, such as EBV+ post-transplantlymphoproliferative diseases (PTLDs).

In another aspect, this disclosure relates to a method of treating orpreventing EBV infection. The method includes administering to a subjectat an elevated risk of EBV infection a therapeutically effective amountof one or more EBV antibodies disclosed herein or an immunogenicfragment thereof, the EBV antibody-small molecule conjugate, one or moreimmunogenic peptides disclosed herein, or the pharmaceutical compositiondescribed above. In some embodiments, the subject is human. In someembodiments, the EBV antibody is a humanized 72A1, or a humanized E1D1.

In another aspect, this disclosure relates to a method of preventing apost-transplant lymphoproliferative disease (PTLD). PTLD is associatedwith EBV infection of B cells, either as a consequence of reactivationof the virus post transplantation or from primary EBV infection. Themethod includes administering to a subject who is a transplant recipienta prophylactically or therapeutically effective amount of one or moreEBV antibodies disclosed herein or an immunogenic fragment thereof, theEBV antibody-small molecule conjugate, one or more immunogenic peptidesdisclosed herein, or the pharmaceutical composition described above. Theadministration can be before, during, and/or after the transplant. Insome embodiments, the subject is a pediatric transplant recipient who isEBV naïve. In some embodiments, the subject is an adult transplantrecipient. In some embodiments, the subject is human.

In another aspect, this disclosure relates to a method of treating anEBV-associated cancer. The method includes administering to a subjectsuffering from an EBV-associated cancer a prophylactically ortherapeutically effective amount of one or more EBV antibodies disclosedherein or an immunogenic fragment thereof, the EBV antibody-smallmolecule conjugate, one or more immunogenic peptides disclosed herein,or the pharmaceutical composition described above. In some embodiments,the examples of EBV-associated cancer include but are not limited toHodgkin lymphoma, Burkitt lymphoma, gastric cancer, and nasopharyngealcarcinoma. In some embodiments, the subject is human. In someembodiments, the EBV antibody is a humanized 72A1, or a humanized E1D1.

In another aspect, this disclosure relates to a method of immunizing orvaccinating a subject against an EBV infection. The method includesadministering to a subject suffering from an EBV infection atherapeutically effective amount of one or more EBV antibodies disclosedherein or an immunogenic fragment thereof, one or more immunogenicpeptides disclosed herein, or the pharmaceutical composition thereof asdescribed above. In some embodiments, the subject is human.

In another aspect, this disclosure relates to a method of inducing theproduction of neutralizing antibodies against an EBV in a subject. Themethod includes administering to a subject an effective amount of one ormore immunogenic peptides disclosed herein. In some embodiments, thesubject is human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show specificity of the novel anti-gp350 mAbs. FIG. 1A:SDS-PAGE analysis of anti-EBV gp350 antibodies purified from indicatedhybridoma (HB) supernatants. FIG. 1B: ELISA screening of hybridoma (HB)supernatants for anti-gp350-specific antibodies. Soluble EBV gp350protein was used as the target antigen at 0.5 μg/ml. m72A1 at 10 μg/mland KSHV anti-gH/gL (54A1) were used as positive and negative (notshown) controls, respectively. Bound antibodies were detected usingHRP-conjugated anti-mouse IgG (1:2,000). Twenty-three HB clones withELISA signals two times greater than those of 54A1 control wereconsidered to be positive/reactive to gp350. FIG. 1C: Immunoblotanalysis with gp350-transfected stable CHO lysate to determinespecificity of anti-gp350-producing HB supernatants. FIG. 1D: Flowcytometric analysis of surface expression of gp350 protein on gp350expressing CHO cells. Cells were stained with indicated anti-gp350 mAb(1:250), followed by secondary goat anti-mouse conjugated to AF488. FIG.1E: Flow cytometric analysis of HB5, HB17 and HB19.

FIG. 2 shows agarose gel analysis of PCR products of heavy chain ofselect novel anti-gp350 antibodies (HB1, HB4, HB7, HB13, and HB15) andm72A1 was used as a positive control.

FIGS. 3A and 3B show PROMALS3D multiple sequence alignment of VH (FIG.3A) and VL (FIG. 3B) regions of 15 mAbs and nAb-72A1 (SEQ IDNOS:100-131). The highly variable complementarity determining regions(CDR) 1-3, indicated by black boxes, define the antigen bindingspecificity. The conserved framework regions (FR) 1-4 flank the CDRs.Consensus amino acid (AA) are in bold and upper case. Consensuspredicted secondary structure (ss) symbols: alpha-helix: h andbeta-strand: e.

FIGS. 4A-4D show the comparison of murine 72A1 (m72A1) and humanized72A1 (h72A1). FIG. 4A: Sequence comparison of murine (m72A1) andhumanized (h72A1) 72A1. ClustalW alignment of heavy chain (i) and lightchain (ii) variable region amino acid sequences. Murine 72A1 heavy chainand light chain AA sequences are represented by SEQ ID NOs: 178 and 179,respectively, and humanized 72A1 heavy chain and light chain AAsequences are represented by SEQ ID NOs: 180 and 181, respectively.Regions of identical sequence are represented by *. Regions ofsimilarity are represented by :. FIG. 4B: ELISA comparison screening ofm72A1 and h72A1 for anti-gp350-specificity. Soluble EBV gp350 proteinwas used as the target antigen at 0.5 μg/ml. m72A1 and h72A1 wereserially diluted (5-0.062 μg/ml) and 1× phosphate buffered saline (PBS)was used as a negative control (data not shown). Bound h72A1 and m72A1antibodies were detected using HRP-conjugated anti-mouse IgG andanti-human IgG (1:2,000) as relevant. FIG. 4C: ELISA determining thereactivity of humanized 72A1 to murine IgG. Soluble EBV gp350 proteinwas used as the target antigen at 0.5 μg/ml. Plates were incubated with10 μg/ml of m72A1 and h72A1, followed by three washes. Bound antibodieswere detected using HRP-conjugated anti-mouse IgG or anti-human IgG(1:2,000). FIG. 4D: Flow cytometric analysis of m72A1 and h72A1 gp350specificity. CHO wild-type cells and gp350-expressing CHO cells werestained with m72A1 and h72A1, followed by secondary goat anti-mouse oranti-human conjugated to AF488. Unstained cells and cells stained withsecondary goat anti-mouse or anti-human conjugated to AF488 alone wereused as negative controls.

FIGS. 5A-5C show neutralization activity of novel anti-gp350 mAbsagainst EBV-eGFP in Raji cells. FIG. 5A: EBV-eGFP titration in Rajicells to determine optimal dose of infection. FIG. 5B: EBV-eGFP waspre-incubated with 15 indicated serial diluted (12.5-100 μg/ml),maxispin column-purified anti-gp350 mAbs, followed by incubation with10⁵ Raji cells for 48 hours. EBV-eGFP+ cells were enumerated using flowcytometry. Anti-gp350 (m72A1) nAb served as positive control andnon-neutralizing anti-gp350 (2L10) mAb and anti-KSHV gH/gL mAb (54A1)served as negative controls. FIG. 5C: EBV-eGFP was pre-incubated with 7indicated serially diluted (12.5-100 μg/ml) protein G affinitychromatography- and size-exclusion chromatography-purified anti-gp350mAbs, followed by incubation with 10⁵ Raji cells for 48 hours. EBV-eGFP+cells were enumerated using flow cytometry. Anti-gp350 (m72A1 and h72A1)nAbs served as positive controls and anti-KSHV gH/gL mAb (54A1) servedas negative control.

FIGS. 6A-6I show the linear peptide epitope mapping of gp350 of variousantibodies. ELISA was used to detect the responses of the indicatedantibodies against each linear peptide. ELISA plates were coatedovernight with 10 μg/ml of each of the indicated 45 linear peptides and0.5 μg/ml of recombinant purified gp350 protein was used as a positivecontrol. Plates were blocked for 1 hour, washed three times, andincubated with 10 μg/ml of each antibody for 2 hours. Bound antibodieswere detected using HRP-conjugated anti-mouse IgG or anti-human IgG(1:2,000).

FIGS. 7A-7B show the schematic diagram (FIG. 7A) and the amino acidsequence (FIG. 7B, SEQ ID NO:182) of EBV gp350 protein, illustrating theectodomain and the splice site (AA 501-699) for making gp220, thetransmembrane domain, TM (AA 841-897) and the cytoplasmic domain, CT (AA898-907). To analyze and classify binding of anti-gp350 mAbs to linearepitopes on the protein, EBV gp350 was separated into 9 regions of ˜100AA.

FIGS. 8A-8C show construction of chimeric gp350 nAbs. FIG. 8A:Construction of chimeric Ab. FIG. 8B: Example of the cloning strategy ofheavy chain and light chain variable regions into expression vectors.FIG. 8C: PCR amplification of heavy chain and light chain variableregions. 72A1 and clone 19 were used as examples of PCR amplification.

FIG. 9 illustrates an antibody-L₂P₄ conjugate.

FIGS. 10A-10B show the comparison of murine E1D1 (mE1D1) and humanizedE1D1 (hE1D1). FIG. 10A: Sequence comparison of mE1D1 and hE1D1.Alignment of heavy chain (i) and light chain (ii) variable region aminoacid sequences by the Clustal W. Murine E1D1 heavy chain and light chainAA sequences are represented by SEQ ID NOs: 183 and 184, respectively,and humanized E1D1 heavy chain and light chain AA sequences arerepresented by SEQ ID NOs: 185 and 186, respectively. Regions ofidentical sequence are represented by *. Regions of similarity arerepresented by :. FIG. 10B: Flow cytometric analysis of mE1D1 and hE1D1gH/gL specificity. CHO wild type cells and gH/gL-expressing CHO cellswere stained with mE1 D1 and hE1 D1, followed by secondary goatanti-mouse or anti-human conjugated to AF488. Unstained cells and cellsstained with secondary goat anti-mouse or anti-human conjugated to AF488alone, were used as negative controls.

FIGS. 11A-11F demonstrate biochemical characterization of chimeric andhumanized antibodies. FIG. 11A is a schematic diagram of murine (m),chimeric (ch), humanized (h) and human antibodies. FIG. 11B showsSDS-PAGE analysis of antibodies under reducing conditions afterpurification using size exclusion chromatography. FIG. 11C shows ELISAdetermining the reactivity of chimeric and humanized antibodies tomurine IgG. FIG. 11D shows Western blot analysis of anti-gp350antibodies detecting liner epitopes. FIG. 11E shows ELISA binding of (i)anti-gp350 and (ii) anti-gH/gL to soluble gp350 and gH/gL proteins. FIG.11F shows flow cytometric analysis of (i) gp350 and (ii) gH/gLspecificity.

FIGS. 12A-12C show the EBV inhibitory effect of anti-gp350 andanti-gH/gL neutralizing antibodies in epithelial and B cells in vitro.Neutralization activity of single and combination anti-gp350 andanti-gH/gL in HEK 293 cells (12A), SVKCR2 cells (12B) and Raji cells(12C). Black dotted line represents 50% neutralization activity.

DETAILED DESCRIPTION

Epstein-Barr virus (EBV) predominantly infects epithelial cells and Bcells, reflecting the viral tropism and cellular ontogeny characteristicof most EBV-associated malignancies (1). Despite the fact that EBVinfection is associated with more than 200,000 cases of a variety ofhuman malignancies every year, and has significant public healthimpacts, there is no licensed vaccine to date (20). The EBV glycoproteingp350/220 (gp350) is a known target for a host's virus neutralizingantibody (nAb) response upon natural EBV infection (52, 67, 71) orimmunization, and thus has been tested as a viable target for vaccinesand therapeutics in five clinical trials to prevent B cell infection(30, 32-35). However, not all of the potential nAb epitopes on gp350have been identified or fully characterized.

EBV infects at least 90% of the human population globally, irrespectiveof geographical location. Currently, there are two models describing howinitial EBV infection of human host cells occurs in vivo (72). In thefirst infection model, the incoming virus first targets epithelial cellsand engages with host ephrin receptor tyrosine kinase A2 viaheterodimeric glycoproteins gH/gL (73, 74) or with host integrins viaBMRF-2 (75, 76). This triggers fusion of EBV glycoprotein gB with thehost epithelial cell membrane to enhance viral entry into the cytoplasm.This interaction is thought to occur in the oral mucosa; there, EBVundergoes lytic replication in epithelial cells to release virions thatsubsequently infect resting B cells in tonsillar crypts or circulatingnaïve B cells. In the alternative infection model, the incoming virusbinds to B cells in the oral mucosa via host CD35 (45) and/or CD21through its major immunodominant glycoprotein, gp350 (55, 65). Theinteraction between gp350 and CD35 and/or CD21 triggers viraladsorption, capping, and endocytosis into B cells (66). Thissubsequently leads to the heterotrimeric EBV glycoprotein complexgp42/gH/gL binding to host HLA class II molecules to activate gBmembrane fusion and infection of B cells (17). Once infected, B cellstypically remain latent and harbor the virus for life, but may alsotraffic back to the oropharynx, where EBV is amplified by lyticreplication in epithelial cells, and shed into the saliva (72). Thus, Bcells are the main reservoirs for EBV reactivation and for thedevelopment of virus-related malignancies (77). Novel strategies thatcould block interactions between EBV glycoproteins and cellularreceptors that mediate viral infection could be beneficial in thedevelopment of effective antiviral therapies.

Antibodies are the first line of defense against viral infection andnearly all EBV-infected individuals develop nAbs directed to theectodomain of EBV gp350 (52, 67, 71). A recent study showed thatpolyclonal serum antibodies against gp350 from naturally infectedindividuals or immunized animals block EBV infection of B cells in vitrobetter than antibodies against EBV gH/gL or gp42 (78). Thus, gp350 is apromising candidate for development of EBV vaccines against B cellinfection; however, to make effective vaccines, the nAbs epitopes on thegp350 ectodomain must be identified and fully characterized.

Disclosed herein are EBV antibodies or immunogenic fragments thereofthat specifically bind to gp350/gp220, immunogenic peptides, and EBVantibody-small molecule conjugates for treating or preventing EBVinfection, in particular, in subjects receiving a transplant. In someembodiments, chimeric (human/mouse) anti-gp350 nAbs or humanizedantibodies or functional fragments thereof are conjugated to L₂P₄ toobtain an EBV-specific ADC that improves the therapeutic efficacy oftreating EBV-associated PTLDs. L₂P₄ described by Jiang et al., NatureBiomedical Engineering 1: 0042 (2017), is an example of the smallmolecules encompassed by this disclosure.

The term “antibody” as used herein refers to an immunoglobulin moleculeor an immunologically active portion thereof that specifically binds to,or is immunologically reactive with a particular antigen, for example,EBV gp350/gp220, or a particular domain or fragment of gp350/gp220 suchas AA1-101, 102-201, and 402-501.

In certain embodiments an antibody for use in the present methods orcompositions is a full-length immunoglobulin molecule, which comprisestwo heavy chains and two light chains, with each heavy and light chaincontaining three complementary determining regions (CDRs). The CDRs ofvarious antibodies are identified and listed in Table 1 below.

TABLE 1 CDR Sequences Anti- VH VL bodies CDR-1 CDR-2 CDR-3 CDR-1 CDR-2CDR-3 m72A1 GSSFTDY INPYNGG GGLRRVNWFAYW TGAVTTSNY GTN VLWHSNHWV(SEQ ID NO: 4) (SEQ ID NO: 20) (SEQ ID NO: 36) (SEQ ID NO: 52)(SEQ ID NO: 68) (SEQ ID NO: 84) h72A1 GSSFTDY INPYNGG GGLRRVNWFAYWTGAVTTSNY GTN VLWHSNHWV (SEQ ID NO: 4) (SEQ ID NO: 20) (SEQ ID NO: 36)(SEQ ID NO: 52) (SEQ ID NO: 68) (SEQ ID NO: 84) HB-1 GFLLTTY IWAGGSRDRGYGYLYAMDYW QNVGTN STD QQYNTYPYT (SEQ ID NO: 5) (SEQ ID NO: 21)(SEQ ID NO: 37) (SEQ ID NO: 53) (SEQ ID NO: 69) (SEQ ID NO: 85) HB-2GYTFTAY INYKTGE PYGYALDYW SSVNY* ATS* QQWSSNPPT* (SEQ ID NO: 6)(SEQ ID NO: 22) (SEQ ID NO: 38) (SEQ ID NO: 54) (SEQ ID NO: 70)(SEQ ID NO: 86) HB-3 GYTFASY INPNNGH* RNLYYYGRPDYW* QDIGNY* YTS*QQGNTLPPT* (SEQ ID NO: 7) (SEQ ID NO: 23) (SEQ ID NO: 39)(SEQ ID NO: 55) (SEQ ID NO: 71) (SEQ ID NO: 87) HB-5 GYTFTNH INPYNDYRSEGWLRRGAWFAY QSIGTS YAS QQSNSWPMLT (SEQ ID NO: 8) (SEQ ID NO: 24)(SEQ ID NO: 40) (SEQ ID NO: 56) (SEQ ID NO: 72) (SEQ ID NO: 88) HB-6GYTFTDY* INTRTGE PYGYALDYW SSVNY* ATS* QQWSSNPPT* (SEQ ID NO: 9)(SEQ ID NO: 25) (SEQ ID NO: 41) (SEQ ID NO: 57) (SEQ ID NO: 73)(SEQ ID NO: 89) HB-7 GYTFTDY* ISPGRSG RYGHPSYLDVW QSVGNA SAS QQYSSYPLT(SEQ ID NO: 10) (SEQ ID NO: 26) (SEQ ID NO: 42) (SEQ ID NO: 58)(SEQ ID NO: 74) (SEQ ID NO: 90) HB-8 GYSFTNY* INTYTGE RYYYGSVYSAWFAYWQSIVHSNGNTY* KVS* FQGSHVPYT* (SEQ ID NO: 11) (SEQ ID NO: 27)(SEQ ID NO: 43) (SEQ ID NO: 59) (SEQ ID NO: 75) (SEQ ID NO: 91) HB-9GFTFSSY ISSGGSY REDFYYGSSYGFFDVW QSIVHSNGNTY* KVS* FQGSHVPYT*(SEQ ID NO: 12) (SEQ ID NO: 28) (SEQ ID NO: 44) (SEQ ID NO: 60)(SEQ ID NO: 76) (SEQ ID NO: 92) HB-10 GYTFTSY* INPSNGH RNLYYYGRPDYWQDIGNY* YTS* QQNTLPPT (SEQ ID NO: 13) (SEQ ID NO: 29) (SEQ ID NO: 45)(SEQ ID NO: 61) (SEQ ID NO: 77) (SEQ ID NO: 93) HB-11 GDSITSG ISYSGSRGNGGNYDWYFDVW SSVNF YIS QQFTSSPSWT (SEQ ID NO: 14) (SEQ ID NO: 30)(SEQ ID NO: 46) (SEQ ID NO: 62) (SEQ ID NO: 78) (SEQ ID NO: 94) HB-12GYTFTNY* INPNNGH* RNLYYYGRPDYW* QSLVHSNGNTY KVS* SQSTHVPLT(SEQ ID NO: 15) (SEQ ID NO: 31) (SEQ ID NO: 47) (SEQ ID NO: 63)(SEQ ID NO: 79) (SEQ ID NO: 95) HB-14 GYTFTDY* IHPRRGG RYGYPWYFDVWQSIVHDNGNTY KVS* FQGSHVPPT (SEQ ID NO: 16) (SEQ ID NO: 32)(SEQ ID NO: 48) (SEQ ID NO: 64) (SEQ ID NO: 80) (SEQ ID NO: 96) HB-17GYTFTSY* INPNNGH* RNLFYYSRPDYW QDIGNY* YTS* QQGNTLPPT* (SEQ ID NO: 17)(SEQ ID NO: 33) (SEQ ID NO: 49) (SEQ ID NO: 65) (SEQ ID NO: 81)(SEQ ID NO: 97) HB-20 GYTFTSY* INPTNGH RNLYYYGRPDYW* QDIGNY* YTS*QQGNALPPT (SEQ ID NO: 18) (SEQ ID NO: 34) (SEQ ID NO: 50)(SEQ ID NO: 66) (SEQ ID NO: 82) (SEQ ID NO: 98) HB-22 GFSLTNY IWSDGSRNYYGNSYPAWFAYW QSIVHSNGNTY KVS* FQGSHVPWT (SEQ ID NO: 19)(SEQ ID NO: 35) (SEQ ID NO: 51) (SEQ ID NO: 67) (SEQ ID NO: 83)(SEQ ID NO: 99)

The term “antibody,” in addition to natural antibodies, also includesgenetically engineered or otherwise modified forms of immunoglobulins,such as synthetic antibodies, intrabodies, chimeric antibodies, fullyhuman antibodies, humanized antibodies, peptibodies and heteroconjugateantibodies (e.g., bispecific antibodies, multispecific antibodies,dual-specific antibodies, anti-idiotypic antibodies, diabodies,triabodies, and tetrabodies). For example, humanized bispecific nAbscomprising E1 D1 and 72A1 targeting two EBV gps, gp350 and gH/gLcomplex, respectively, can be produced for use as a prophylactic agentagainst EBV infection or re-infection. In some embodiments, bispecificor multispecific antibodies comprising a combination of the mAbsidentified herein or immunogenic fragments thereof can be produced. Thebispecific or multispecific antibodies can be humanized according toknown technologies. Alternatively, the bispecific or multispecificantibodies can be chimeric antibodies. The antibodies disclosed hereincan be monoclonal antibodies or polyclonal antibodies. In thoseembodiments wherein an antibody is an immunologically active portion ofan immunoglobulin molecule, the antibody may be, for example, a Fab,Fab′, Fv, Fab′ F(ab′)2, disulfide-linked Fv, single chain Fv antibody(scFv), single domain antibody (dAb), or diabody. The antibodiesdisclosed herein, including those that are immunologically activeportion of an immunoglobulin molecule, retain the ability to bind aspecific antigen such as EBV gp350/220, or to bind a specific fragmentof gp350/gp220 such as AA1-101, AA102-201, and AA402-501.

In some embodiments, the EBV antibodies disclosed herein have undergonepost-translational modifications such as phosphorylation, methylation,acetylation, ubiquitination, nitrosylation, glycosylation, or lipidationassociated with expression in a mammalian cell line, including a humanor a non-human host cell. Techniques for producing recombinantantibodies and for in vitro and in vivo modifications of recombinantantibodies are known in the art.

Provided in certain embodiments herein are chimeric, and/or humanizedEBV antibodies. Various techniques are known in the art for humanizingantibodies from non-human species such that the antibodies are modifiedto increase their similarity to antibodies naturally occurring inhumans. Six CDRs are present in each antigen binding domain of a naturalantibody. These CDRs are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding domain asthe antibody assumes its three-dimensional configuration. CDR sequencesof certain antibodies identified herein are shown in Table 1. Theremainder of the amino acids in the antigen binding domains, referred toas “framework” regions, show less inter-molecular variability and form ascaffold to allow correct positioning of the CDRs. This disclosure alsorelates to antibodies comprising VH and VL regions comprising the CDRsshown in Table 1.

“Treating” or “treatment” of a disease or a condition may refer topreventing the disease or condition, slowing the onset or rate ofdevelopment of the disease or condition, reducing the risk of developingthe disease or condition, preventing or delaying the development ofsymptoms associated with the disease or condition, reducing or endingsymptoms associated with the disease or condition, generating a completeor partial regression of the disease or condition, or some combinationsthereof.

As used herein, the term “subject” refers to mammalian subject,preferably a human. The phrases “subject” and “patient” are usedinterchangeably herein.

The method for treating a condition or a viral infection includesadministering a therapeutically effective amount of a therapeutic agentor a pharmaceutical composition. An “effective amount,” “therapeuticallyeffective amount” or “effective dose” is an amount of a composition(e.g., a therapeutic agent or a pharmaceutical composition) thatproduces a desired therapeutic effect in a subject, such as preventingor treating a target disease or condition, or alleviating symptomsassociated with the disease or condition. The precise therapeuticallyeffective amount is an amount of the composition that will yield themost effective results in terms of efficacy of treatment in a givensubject. This amount will vary depending upon a variety of factors,including but not limited to the characteristics of the therapeuticagent (including activity, pharmacokinetics, pharmacodynamics, andbioavailability), the physiological condition of the subject (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage, and type of medication), the nature ofthe pharmaceutically acceptable carrier or carriers in the formulation,and the route of administration. One skilled in the clinical andpharmacological arts will be able to determine a therapeuticallyeffective amount through routine experimentation, namely by monitoring asubject's response to administration of a compound and adjusting thedosage accordingly. For additional guidance, see Remington: The Scienceand Practice of Pharmacy 21^(st) Edition, Univ. of Sciences inPhiladelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa.,2005.

The pharmaceutical composition may include, among other things, one ormore EBV antibodies disclosed herein or one or more immunogenicfragments thereof, one or more immunogenic peptides disclosed herein, oran EBV antibody-small molecule conjugate disclosed herein.

The pharmaceutical composition may also include one or morepharmaceutically acceptable carriers. A “pharmaceutically acceptablecarrier” refers to a pharmaceutically acceptable material, composition,or vehicle that is involved in carrying or transporting a compound ofinterest from one tissue, organ, or portion of the body to anothertissue, organ, or portion of the body. For example, the carrier may be aliquid or solid filler, diluent, excipient, solvent, or encapsulatingmaterial, or some combination thereof. Each component of the carriermust be “pharmaceutically acceptable” in that it must be compatible withthe other ingredients of the formulation. It also must be suitable forcontact with any tissue, organ, or portion of the body that it mayencounter, meaning that it must not carry a risk of toxicity,irritation, allergic response, immunogenicity, or any other complicationthat excessively outweighs its therapeutic benefits.

The pharmaceutical compositions described herein may be administered byany suitable route of administration. A route of administration mayrefer to any administration pathway known in the art, including but notlimited to aerosol, enteral, nasal, ophthalmic, oral, parenteral,rectal, transdermal (e.g., topical cream or ointment, patch), orvaginal. “Transdermal” administration may be accomplished using atopical cream or ointment or by means of a transdermal patch.“Parenteral” refers to a route of administration that is generallyassociated with injection, including infraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. In some embodiments, the therapeuticcompositions described herein are administered by intravenous injectionor intraperitoneal injection.

In certain embodiments, disclosed herein is a method of treating orpreventing EBV infection in a subject, comprising administering atherapeutically effective amount of one or more anti-gp350 antibodiesdisclosed herein or one or more immunogenic fragments thereof, one ormore immunogenic peptides described herein, an EBV antibody-drugconjugate described herein, or a pharmaceutical composition comprisingthe anti-gp350 antibody or an immunogenic fragment thereof, one or moreimmunogenic peptides, or the EBV antibody-drug conjugate.

In certain embodiments, disclosed herein is a method of treating orpreventing EBV infection in a subject, comprising administering atherapeutically effective amount of an anti-gp350 antibody disclosedherein or an immunogenic fragment thereof, an immunogenic peptidedescribed herein, an EBV antibody-drug conjugate described herein, or apharmaceutical composition comprising the anti-gp350 antibody or animmunogenic fragment thereof, one or more immunogenic peptides, or theEBV antibody-drug conjugate.

In certain embodiments, disclosed herein is a method of treating orpreventing EBV-associated PTLD in a subject, comprising administering atherapeutically effective amount of an anti-gp350 antibody disclosedherein or an immunogenic fragment thereof, one or more immunogenicpeptides described herein, an EBV antibody-drug conjugate describedherein, or a pharmaceutical composition comprising the anti-gp350antibody or an immunogenic fragment thereof, one or more immunogenicpeptides, or the EBV antibody-drug conjugate, before or after thetransplant in the subject.

As shown in the working examples, 15 novel EBV gp350-specific mAbs weregenerated, their binding to gp350 was characterized, theirneutralization activity against EBV infection in vitro was determined,and their epitopes were mapped. The newly developed mAbs have many usesin vaccine development, diagnosis of viral infection, andtherapeutic/prophylactic management of post-transplantlymphoproliferative diseases, either individually, in combination withnAb-72A1, or with other mAbs such as anti-gH/gL (E1D1).

To overcome the existing challenges facing PTLD treatment, novel EBVantibodies and EBV antibody-drug conjugates (ADCs) are developed. TheEBV neutralizing antibodies (nAbs) that specifically block new orreactivated EBV infection are conjugated with small molecules thatspecifically target latent viral protein, EBV nuclear antigen 1expressed in all EBV+ malignancies. The recent identification andisolation of nAbs against the highly variable viruses HIV-1 (10, 11),influenza (12-14), and respiratory syncytial virus (15) has directimplications for successful EBV protection. Indeed, in 2012, aninternational, multidisciplinary expert panel recommended use ofintravenous (IV) anti-viral nAbs for preventing or treating EBV+ PTLD(16). EBV uses multiple surface glycoproteins (gps), including the majorgp350, to infect host cells (17, 18). These gps are expressed on EBVvirions and in EBV+ cells (19, 20), and stimulate immune responses inhumans and in animal models (21-23), making them attractive targets foran EBV vaccine (24). Multiple lines of evidence suggest that use ofanti-gp350 nAbs to protect against EBV-PTLDs is feasible (16): (A)Maternal Abs protect against EBV infection in neonates (25, 26); (B)gp350-expressing EBV+ cells activate complement (27) and mediateAb-dependent cellular cytotoxicity (28); (C) gp350 vaccines reduce EBVload and protect against EBV+ lymphomas in marmosets (29-32) and protectEBV-naïve adults from EBV-induced mononucleosis (32-34); (D) Compared tocontrol mice, SCID mice injected with peripheral blood mononuclear cellsfrom EBV-naïve donors and immunized with anti-gp350 (72A1) mouse nAb arecompletely protected against EBV and development of EBV+tumors orPTLD-like lesions (35); and (E) 72A1 also conferred short-termprotection against acquiring EBV after transplantation in 3 out of 4pediatric patients in a small phase 1 clinical trial (35). However,there was a major drawback: all 4 patients who received 72A1 developedhuman anti-mouse Abs (HAMA), which can cause side effects and limittreatment efficacy, with one developing a hypersensitivity reaction.This suggests that 72A1 in its native form is not a safe treatment forhumans (35). Thus, chimeric (human/mouse), humanized, or human nAbs,which are safe and effective in the treatment of various cancers, areneeded (7, 36-38).

Pre-existing antibodies provide the primary defense against viralinfection. Prophylactic prevention of EBV primary infection has mainlyfocused on blocking the first step of viral entry by generatingneutralizing antibodies (nAbs) that target EBV envelope glycoproteins.Five glycoproteins, in particular, gp350/220 (gp350), gp42, gH, gL, andgB, are required for efficient infection of permissible host cells andhave emerged as potential prophylactic targets (23, 24, 61, 64).

Several studies have indicated that the EBV gp350 as the majorimmunodominant glycoprotein is an ideal target for EBV nAbs production.Although the ectodomain of EBV gp350 (AA 1-841) has been shown tocontains at least seven unique CD21 binding epitopes located in theectodomain of gp350 (58), at least one of these epitopes (AA 142-161) iscapable of eliciting nAbs (57-58). The AA residues 142-161 are also oneof the binding epitopes for nAb 72A1 (59, 68). The AA residues thatconstitute the other epitopes and their role in generating nAb has notbeen elucidated, as this information would be valuable in the precisedesign of effective EBV peptide vaccine. To date, nAb-72A1 remains theonly EBV antibody with proven clinical prophylactic efficacy, as itsbeen shown to confer short-term protection by reducing and delaying EBVinfection onset in immunized pediatric transplant patients (35).

EBV predominantly infects epithelial cells and B cells, reflecting theviral tropism and the cellular ontogeny for EBV-associated malignancies(17). There are two schools of thought on how the initial EBVtransmission into the human host occurs. In the first infection model,the incoming virus engages with ephrin receptor A2 via heterodimericgH/gL, which triggers gB fusion with the epithelial cell membrane andentry of the virus into the cytoplasm (17). This interaction is thoughtto occur in the oral mucosa, where the virus undergoes lytic replicationto release virions that subsequently infect B cells. In the alternativemodel, the incoming virus binds to the host cell via complement receptortype 1 (CR1)/CD35 (45) and/or CR2/CD21 through its major immunodominantglycoprotein, gp350 (65). The interaction between gp350 and CD35 and/orCD21 triggers viral adsorption, capping, and endocytosis into the B cell(66), which subsequently leads to interaction between heterotrimericviral glycoproteins complex, gp42/gH/gL, binding to HLA class IImolecules to activate gB membrane fusion and entry. Because these twomodels are not necessarily mutually exclusive, and given that both gp350and gH/gL complex are important in initiating the first viral contactwith host cells, use of nAbs that target either gp350 or gH/gL complexor both may potently block incoming virus at the oral mucosa.

Nearly all EBV-infected individuals develop nAbs directed to theectodomains of these glycoproteins (52, 67). These antibodies canprevent neonatal infection, and can protect against acute infectiousmononucleosis in adolescents and several human lymphoid and epithelialmalignancies associated with EBV infection (30, 32-34). Althoughnumerous monoclonal antibodies (mAbs) have been generated against EBVgp350 (53, 68, 69), only two murine mAbs, the non-neutralizing 2L10 andthe neutralizing 72A1, have been extensively characterized and madecommercially available (68, 69). Importantly, nAb-72A1 conferredshort-term clinical protection against EBV transmission aftertransplantation in pediatric patients in a small phase I clinical trial(35).

EBV gp350 is the most immunogenic envelope glycoproteins on the virions.It is a type 1 membrane protein that encodes for 907 amino acidresidues. A single splice of the primary transcript deletes 197 codonsbetween codons 501 and 699, and joins two fragments of gp350 codons inframe to generate the gp220 messenger RNA. Both gp350 and gp220 arecomprised of the same 18-AA residue at the C terminus that is locatedwithin the viral membrane, a 25-AA residue at the transmembrane-spanningdomain, and a large highly glycosylated N-terminal ectodomain, AA 1-841(57). The first 470 AA of gp350 are sufficient for binding CD21 in Bcells, as demonstrated by a truncated gp350 (AA 1-470) blocking thebinding of EBV to B cells and reducing viral infectivity (17). Thegp350-binding domain on CD21 is mapped to N-terminal short consensusrepeats (SCRs) 1 and 2, which also bind to a bioactive fragment ofcomplement protein 3 (C3d) (34, 68). A soluble truncated EBV gp350fragment (AA 1-470) and soluble CD21 SCR1 and SCR2 can block EBVinfection and immortalization of primary B cells (57). However, gp350binding to CD35 is not restricted to N-terminal SCRs; it binds longhomologous repeat regions as well as SCRs 29-30 (57).

The gp350 ectodomain is heavily glycosylated, with both N- and O-linkedsugars, which accounts for over half of the molecular mass of theprotein. Currently there is only one crystal structure available forgp350, comprised of a truncated structure between 4-443 AA, with atleast 14 glycosylated asparagine residues coating the protein withsugars, with the exception of a single glycan-free patch (59).Mutational studies of several residues in the glycan-free patch resultedin the loss of CD21 binding (59), suggesting that binding of CD35 andCD21 by gp350 is mediated within this region.

Using gp350 synthetic peptides binding to CD21 on the surface of a Bcell line, additional gp350 epitope was identified in the C-terminalregion of gp350 (AA 822-841), suggesting that this region is involved inEBV infection of B cells (58). The role of other epitopes in elicitingnAbs has not been fully investigated. Furthermore, the exact AA residuesthat comprise the core binding epitopes capable of elicitingneutralization and non-neutralization antibodies have not beendetermined. Mapping the EBV gp350 protein residues definingimmunodominant epitopes, identifying the critical AA residues of theepitopes, and defining their roles in generating nAbs and non-nAbs willguide rational design and construction of an efficacious EBV gp350-basedvaccine that would focus the B-cell responses to the protectiveepitopes.

As demonstrated in the working examples, 23 hybridomas producingantibodies against EBV gp350 were generated. To assess their clinicaland diagnostic potential and utility in informing future prophylacticand therapeutic vaccine design: (1) the ability of the antibodiesproduced by the new hybridomas to detect gp350 protein was tested byenzyme-linked immunosorbent assay (ELISA), flow cytometry, andimmunoblot; (2) the unique CDRs of the heavy and light chains of all 23hybridomas were sequenced to identify novel mAbs; (3) the efficacy ofeach mAb to neutralize EBV infection in vitro was measured; and (4)competitive cell and/or linear peptide binding assays were used toidentify gp350 regions recognized by neutralizing and non-neutralizingmAbs; and (5) peptide binding assays were used to identify gp350 corelinear AA residues recognized by neutralizing and non-neutralizing mAbs.

Out of the 23 hybridomas, 15 were monoclonal and novel, based on theirVH and VL CDR sequences, compared to the reported sequence of m72A1(50). Following confirmation that the new 15 mAbs recognized gp350antigen and contained unique VH-VL sequences, further characterizationrevealed that mAbs HB1, HB5, HB11, and HB20 inhibited EBV infection of ahuman B-cell line in a dose-dependent manner, with HB5 being the bestneutralizer, comparable to m72A1 and h72A1. Thus, provided herein arefour new nAbs against EBV infection of B cells with potential clinicalutility in blocking viral infection in immunosuppression settings. Theamino acid sequences of the heavy chain and light chain of the mAbs areprovided in Table 2 below.

TABLE 2 Sequences of Heavy Chain and Light Chain Heavy Chain Light Chain72A1 PELVKPGTSMKISCKASGSSFTDY QAVLTQESALTTSPGETVTLTCRSSTTMNWMKQSHGKNLEWIGLINPYNG GAVTTSNYANWVQEKPDHLFTGLIGGGTRYNQKFKGKATLTLDKSSSTAY TNNRVPGVPARFSGSLIGDKAALTITMEVLSLTSEDSAVYYCAGGLRRVN GAQTEDEAIYFCVLWHSNHWVFGGGTWFAYWGQGTLVSVSAAKTTPPSVY KLTVL (SEQ ID NO: 101) PLAPGSAAQTNSMVTLG (SEQID NO: 100) HB1 PGLVAPSQSLSITCTVSGFLLTTY KFMSTSVGDRVSVTCKASQNVGTNVAGVHWVRQPPGKGLEWLGVIWAGGS WYQQKPGQSPKALIYSTSSRYTGVPDTNYNSALMSRLSINKDISKSQVFL RFAGSGSGTDYTLTISNVQSEDLAEYKMNSLQTDDTAMYYCTRDRGYGYL FCQQYNTYPYTFGGGTRLDIKRADAAYAMDYWGQGTSVTVSSAKTTPPSV PTV (SEQ ID NO: 103) YPLAPGSAAQTNSMVTLG (SEQID NO: 102) HB2 PELKKPGETVKISCKASGYTFTAY AILSASPGEKVTMTCRATSSVNYMHWSMHWVKLTPGKGLKWMGWINTKTG YQQKPGSSPKPWIYATSNLASGVPAREPTYADDFKGRFAFSLETSASTAY FSGSGSGTSYSLTISRVEAEDAATYYLQINNLKNEDTATYFCAPYGYALD CQQWSSNPPTFGAGTKLELKRADAAPYWGQGTSVTVSSAKTTPPSVYPLA TV (SEQ ID NO: 105) GPSAAQTNSMVTLG (SEQ IDNO: 104) HB3 AELVRPGASVKLSCKASGYTFASY SSLSASLGDRVTISCRASQDIGNYLNWMQWVKQWPGQGLEWIGEINPNNG WYQQKPDGTIKLLIYYTSRLHSGVPSHTNYNERFKNKASLTVDKSSSTAY RFSGSGSGTDYSLTISNLEEEDIATYMQLSSLTSEDSAVYYCARNLYYYG FCQQGNTLPPTFGGGTKLEIKRADAARPDYWGQGTSVTVSSAKTTAPSVY PTV (SEQ ID NO: 107) PLAPVCGDTTGSSVTLG (SEQID NO: 106) HB5 AELVRPGASVKISCKAFGYTFTNH AILSVSPGERVSFSCRASQSIGTSIHNINWVKQRPGQGLDWIGYINPYND WYQQRTNDSPRLLIKYASESISGIPPYTSYNQKFKGKATLTVDKSSNTAY RFSGSGSGTDFTLSINSVESEDIADYMELSSLTSEDSAVYYCARSEGWLR HCQQSNSWPMLTFGAGTKLELKRADARGAWFAYWGQGTLVTVSAAKTTAP APTV (SEQ ID NO: 109) SVYPLAPVCGDTTGSSVTLG(SEQ ID NO: 108) HB6 PELRKPGETVKISCKASGYTFTDY AILSASPGEKVTMTCRATSSVNYMHWSMHWVKQTPGKGLKWMGWINTRTG YQQKPGSSPKPWIYATSNLASGVPAREPRYADDFKGRFAFSLETSASTAY FSGSGSGTSYSLTISRVEAEDAATYYLQINNLKNEDTATYFCAPYGYALD CQQWSSNPPTFGAGTKLELKRADAAPYWGQGTSVTVSSAKTTPPSVYPLA TV (SEQ ID NO: 111) PGSAAQTNSMVTLG (SEQ IDNO: 110) HB7 AELVRPGASVKLSCKALGYTFTDY KFMSTSVGDRVNITCKASQSVGNAVAEMHWVKQTPVHGLEWIGTISPGRS WFQQKPGQSPKLLIYSASNRYTGIPDGTAYNQKFKGKATLTADKSSRTAY RFTGSGSGTDFTLTCNNMQSEDLADYMELNSLTSEDSAVYYCSRYGHPSY FCQQYSSYPLTFGAGTKLELKRADAALDVWGAGTTVTVSSAKTTPPSVYP PTV (SEQ ID NO: 113) LAPGSAAQTNSMVTLG (SEQ IDNO: 112) HB8 PELKKPGETVKISCKASGYSFTNY LSLPVSLGDQASISCRSSQSIVHSNGGMNWVKQAPGKGLKWMGWINTYTG NTYLEWYLQKAGQSPKLLIYKVSNRFEPTYADDFKGRFAFSLETSASTAF SGVPDRFGGSGSGTDFTLKISRVEAELQINNLKNEDTATYLCARYYYGSV DLGVYYCFQGSHVPYTFGGGTKLEIKYSAWFAYWGQGTLVTVSAAKTTPP RADAAPTV (SEQ ID NO: 115) SVYPLAPGSAAQTNSMVTLG(SEQ ID NO: 114) HB9 GGLVKPGGSLKLSCAASGFTFSSY LSLPVSLGDQASISCRSSQSIVHSNGTMSWVRQTPEKRLEWVATISSGGS NTYLEWYLQKAGQSPKLLIYKVSNRFYIYYPDSVKGRFTISRDNAKNTLY SGVPDRFGGSGSGTDFTLKISRVEAELQMSSLKSEDTAIYYCTREDFYYG DLGVYYCFQGSHVPYTFGGGTKLEIKSSYGFFDVWGAGTTVTVSSAKTTA RADAAPTV (SEQ ID NO: 117) PSVYPLAPVCGDTTGSSVTLG(SEQ ID NO: 116) HB10 AELVRPGASVKLSCKASGYTFTSYSSLSASLGDRVTISCRASQDIGNYLN WMHWVKQWPGQGPEWIGEINPSNGWYQQKPDGTVKLLIYYTSRLHSGVPS HTNYNERFKNKATLTVDKSSSTAYRFSGSGSGTDYSLTISNLEEEDIATY MQLSSLTSEDSAVYYCARNLYYYGFCQQGNTLPPTFGGGTKLEIKRADAA RPDYWGQGTSVTVSSAKTTPPSVY PTV (SEQ ID NO: 119)PLAPGSAAQTNSMVTLG (SEQ ID NO: 118) HB11 PSLVKPSQTLSLTCSVTGDSITSGAIMSASLGEKVTMSCRASSSVNFMNW FWNWIRKFPGNKLEYMGYISYSGSYQQKSDDSPKLLIYYISNLAPGVPAR TYYNPSLKSRISITRDTSKNQYYLFSGSGSGNSYSLTISGMEGEDAATYY QLNSVTTEDTATYYCARGNGGNYDCQQFTSSPSWTFGGGTKLEIKRADAA WYFDVWGAGTTVTVSSAKTTPPSV PTV (SEQ ID NO: 121)YPLAPGSAAQTNSMVTLG (SEQ ID NO: 120) HB12 AELVRPGASVKLSCKASGYTFTNYLSLPVSLGDQASISCRSSQSLVHSNG WIHWVKQWPGQGLEWIGEINPNNGNTYLHWYLQKPGQSPKLLIYKVSNRF HTNYNERFKNKASLTVDKSSSTAYSGVPDRFSGSGSGTDFTLKISRVEAE MQLSSLTSEDSAVYYCARNLYYYGDLGVYFCSQSTHVPLTFGSGTKLEIK RPDYWGQGTSVTVSS (SEQ ID (SEQ ID NO: 123)NO: 122) HB14 SGAELVRPGASVNLSCKALGYTFT LSLPVSLGDQASISCRSSQSIVHDNGDYEMHWVKQTPVYGLEWIGTIHPR NTYLEWYLQKPGQSPKLLIYKVSNRFRGGTAYNQRFKGKAALTADKSSST SGVLDKFSGSGSGTDFTLKISRVEAEAYMELSSLTSEDSAVYYCARYGYP DLGIYYCFQGSHVPPTFGGGTKLEIKWYFDVWGAGTTVTVSS (SEQ ID (SEQ ID NO: 125) NO: 124) HB17AELVIPGASVKVSCKASGYTFTSY SSLSASLGDRVTISCRASQDIGNYLNWIHWVKQWPGQGLEWIGEINPNNG WYQQKPDGTIKLLIYYTSRLHSGVPSHTNYNEKFKSKATLTVDKSSSTAY RFSGSGSGTDYSLTISNLEEEDIATYMQLSSLTSEDSAVYYCARNLFYYS FCQQGNTLPPTFGGGTKLEIKRADAARPDYWGQGTSVTVSSAKTTPPSVY PTV (SEQ ID NO: 127) PLAPGCGDTTGSSVTLG (SEQID NO: 126) HB20 AELVKPGASVKLSCKASGYTFTSY SSLSASLGDRVTISCRASQDIGNYLNWIQWVKQRPGQGLEWIGEINPTNG WYQQKPDGTVKLLIYYTSRLHSGVPSHTNYNEKFKTKATLTVDKSSSTAY RFSGSGSGTDYSLTISNLEQEDIATYMRLSSLTSEDSAVYYCARNLYYYG FCQQGNALPPTFGGGTKLEIKRADAARPDYWGQGTSVTVSSAKTTAPSVY PTV (SEQ ID NO: 129) PLAPVCGDTTGSSVTLG (SEQID NO: 128) HB22 PGLVAPSQSLSITCTVSGFSLTNY LSLPVSLGDQASISCRSSQSIVHSNGGIHWVRQPPGKGLEWLVVIWSDGS NTYLEWYLQKPGQSPKLLIYKVSNRFTIYNSALKSRLSISKDNSKSQVFL SGVPDRFSGSGSGTDFTLRISRVEAEKMNSLQTDDTAMYYCARNYYGNSY DLGVYYCFQGSHVPWTFGGGTKLEIKPAWFAYWGQGTLVTVSAAKTTPPS RADAAPTV (SEQ ID NO: 131) VYPLAPGSAAQTNSMVTLG(SEQ ID NO: 130)

The identification of four new potent nAbs (HB1, HB5, HB11 and HB20) andsequencing of their CDR regions, as well as the humanization of m72A1and E1D1, opens the possibility of using these nAbs for clinicalapplications, such as reducing or preventing EBV infection in transplantsettings, with the consequent potential to reduce the incidence of EBV+PTLDs. The h72A1 IgG1 antibody recognized both native gp350 as well asgp350 peptides that constitute the principal gp350 neutralizing epitope(142-161) and completely eliminated anti-murine IgG immunoreactivity.Importantly, h72A1—and the four newly generated nAbs (HB1, HB5, HB11,and HB20)—significantly blocked in vitro EBV infection of B cells to adegree comparable to or better than m72A1. Combining two or more nAbsthat bind to different peptides on gp350 and gH/gL can significantlyreduce infection in both B cells and epithelial cells.

In addition, both nAbs and non-nAbs can be used as research tools toprovide insight into epitope targets important for vaccine development.In the past, various methods, including lectin/ricin immune-affinityassay, purified mAbs, purified soluble gp350 mutants, syntheticpeptides, cell binding assays, and X-ray crystallography of partialgp350 protein (AA 4-443), have been used to identify the critical gp350epitopes responsible for its interaction with the CD21 and CD35 cellularreceptors (summarized in Table 3). Despite several attempts to identifygp350 epitopes important for eliciting nAbs, to date only a singleepitope, AA 142-161, has been identified, which is also the bindingepitope for nAb 72A1. Currently, the lack of a crystal structure offull-length gp350 protein and the unavailability of multiple nAbs hinderthe opportunity to identify other gp350 epitopes that might elicit nAbsand inform design of effective vaccine strategies. To identify gp350epitopes responsible for eliciting nAbs, the newly generated nAbs (HB1,HB5, HB11, and HB20) and non-nAbs (HB10, HB17, and HB22) were used toperform competitive cell binding and ELISA-based linear peptide bindingassays. Although both approaches have various limitations, they offeruseful information that when combined might inform and/or advancevaccine development efforts. Competitive cell binding assays can provideinformation on whether two antibodies bind overlapping ornon-overlapping epitopes, although they are unable to indicate whetherthe competing antibodies bind the same or nearby epitopes, nor identifyactual AA residues involved in the binding. On the other hand, the ELISApeptide binding assay is only reactive to linear epitopes and may or maynot take into consideration post-translational protein modifications,depending on whether a full protein or peptides are used as the targetantigen(s).

Using biotinylated antibodies, it was shown that the newly generatedgp350 nAbs (HB1, HB5, HB11, and HB20) bound targets that overlapped withthose of both m72A1 and h72A1, although HB20 showed only partial bindingto the overlapping targets. The non-Ab, HB17 showed little to nocompetitive binding when compared to nAbs, suggesting that they bounddifferent gp350 epitopes. These results strongly suggest that twodistinct binding regions have been identified, one bound predominantlyby nAbs and the other by non-nAbs, and that nAbs potentially bindtargets within the same proximity, if not the same AA sequences. Thus,the current antibodies provide the first step toward generating reagentsrequired for mapping neutralizing versus non-neutralizing epitopes ongp350, should the full-length crystal structure of the protein remainunavailable. Using linear peptide epitope mapping, three majormAb-binding regions, 1-101, 102-201, and 402-501 were identified; allthree regions incorporable previously identified linear epitopes (58,51, 56). Regions 1-101 and 402-501 were bound by both nAbs and non-nAbs,suggesting that these regions are immunodominant. However, the 102-201region containing the nAb epitope 142-161 was only bound by nAbs (HB5,HB11, HB20, and both m72A1 and h72A1), with the exception of the nAbHB1. These results suggest that epitopes/regions capable of elicitingnAbs are located within the N-terminus of gp350.

Previous studies have generated and characterized several anti-gp350mAbs, both neutralizing and non-neutralizing. Some of these have beeneffectively used to map the immunodominant or neutralizing epitopespresent in the gp350 ectodomain, which has relevance for futurestrategies to design sterilizing prophylactic vaccines (Table 3).

TABLE 3 Summary of published gp350 epitope mapping using variousmethodologies Method mAbs/protein/peptides Number of epitopes identifiedReference Competitive binding assay mAbs 7 epitopes - Sequence not (53)defined Binding studies: mAbs 2 possible regions identified (54)Determine the effects of by sequence alignment to C3d anti-gp350 mAbs ongp350 sequence: binding to CR2 1. aa 21-28 2. aa 372-378 Peptide digestand Truncated and mutant Narrowed down to the first (57)immunoprecipitation protein; mAbs (72A1 and 470 residues BOS-1) Bindingstudies Peptide and protein 2 sequences defined: (55) 1. aa 21-28 2.N-terminus of gp350 Dot Blot immunoassay: Protein - 8 clones overlapping3 sequences defined: (56) Purified truncated protein N- and C- terminalportions 1. aa 310-325 incubated with mAbs of protein; 2. aa 326-439mAbs from Qualtiere et al., 3. aa 733-841 1987 Peptide cell bindingassay Synthesized peptides 7 regions, 3 identified: (58) to 2CR2-positive (Raji and covering gp350 (907 aa) 1. aa 142-161 Ramos) and1 -negative 2. aa 282-301 (P3HR-1) cell lines 3. aa 822-841 Crystalstructure and Mutant proteins; mAbs 72A1 3 epitopes (based on 72A1 (59)binding studies binding and gp350 4-443) 1. aa 16-29 2. aa 142-161 3. aa282-301 Structural docking studies gp350 and CR2 crystal Single epitope(based on (60) and antigenicity mapping structure alignment/dockinggp350 aa 1-470) 1. aa 147-165 Structural alignment: Peptides (used in 4epitopes: identified (51) computer modeling of immunization); mAb(72A1) 1. aa 14-20 gp350 and 72A1 and 2. aa 144-161 docking studies 3.aa 194-211 4. aa 288-291

Virus-specific treatments are less likely to target basic metabolicmechanisms of healthy cells, making them more likely to efficiently killvirus-infected cells with fewer side effects. Until recently, few drugregimens have specifically targeted EBV+ lymphomas. However, in 2015, afew small molecules showed activity against EBV-transformed cells (39).Furthermore, in 2017, Jiang et al. described a novel small molecule(L₂P₄) that shows discriminating anti-proliferative activities againstEBV-transformed B lymphoma cells (40).

Having described the invention with reference to the embodiments andillustrative examples, those in the art may appreciate modifications tothe invention as described and illustrated that do not depart from thespirit and scope of the invention as disclosed in the specification. Theexamples are set forth to aid in understanding the invention but are notintended to, and should not be construed to limit its scope in any way.The examples do not include detailed descriptions of conventionalmethods. Such methods are well known to those of ordinary skill in theart and are described in numerous publications. Further, all referencescited above and in the examples below are hereby incorporated byreference in their entirety, as if fully set forth herein.

EXAMPLES Example 1: Materials and Methods

Cells and viruses.

AGS-EBV-eGFP, a human gastric carcinoma cell line infected with arecombinant Akata virus expressing enhanced fluorescent green protein(eGFP) was a kind gift of Dr. Lisa Selin (University of MassachusettsMedical School). Anti-EBV gH/gL (E1D1) hybridoma cell line was a kindgift of Dr. Lindsey Hutt-Fletcher (Louisiana State University HealthSciences Center). Chinese hamster ovary cells (CHO); human embryonickidney cells expressing SV-40 T antigen (HEK-293T); HEK-293 6Esuspension cells; EBV-positive Burkitt lymphoma cells (Raji); myelomacells (P3X63Ag8.653); and anti-EBV gp350 nAb-72A1 hybridoma cells(HB168) were purchased from American Type Culture Collection (ATCC).ExpiCHO cells were purchased from ThermoFisher Scientific.

AGS-EBV-eGFP cells were maintained in Ham's F-12 media supplemented with500 μg/ml neomycin (G418, Gibco). Raji, P3X63Ag8.653, and HB168hybridoma cells were maintained in RPMI 1640. CHO and HEK-293T cellswere maintained in DMEM. HEK-293 6E cells and ExpiCHO cells weremaintained in FreeStyle F17 Expression Medium supplemented with 0.1%Pluronic F-68 and Gibco ExpiCHO Expression Media, respectively. Allculture media were supplemented with 10% fetal bovine serum (FBS) fromMillipore Sigma, 2% penicillin-streptomycin, and 1% I-glutamine, withthe exception of Freestyle F17 expression medium and Gibco ExpiCHOExpression Media. All media were purchased from ThermoFisher Scientificunless otherwise specified.

Antibodies and Plasmids.

Primary antibodies: EBV anti-gp350 nAb (m72A1) and anti-gH/gL (E1D1)were purified from the supernatant of HB168 and E1D1 hybridoma celllines, respectively, using Capturem™ Protein A Maxiprep spin columns(Takara) or protein G affinity and size-exclusion chromatography. Thenon-nAb anti-gp350/220 mAb (2L10) was purchased from Millipore Sigma.Anti-KSHV gH/gL 54A1 mAb was generated and characterized as previouslydisclosed (79).

Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goatanti-mouse IgG for immunoblot or ELISA were purchased from Bio-Rad.HRP-conjugated goat anti-human IgG for ELISA was purchased fromThermoFisher Scientific. Alexa Fluor (AF) 488-conjugated goat anti-mouseIgG (H+L) for flow cytometry was purchased from Life Sciences Tech. Goatanti-mouse IgG (H+L) secondary antibody and DyLight 650 for epitopemapping were purchased from Thermo Fisher Scientific. Anti-biotinmonoclonal antibody conjugated to AF488 for competitive binding assaywas purchased from ThermoFisher Scientific.

The construction of the pCI-puro vector and pCAGGS-gp350/220-F has beendescribed (23, 45).

Virus Production and Purification.

eGFP-tagged EBV was produced from the EBV-infected AGS cell line asdescribed (46). Briefly, AGS-EBV-eGFP cells were seeded to 90%confluency in T-75 flasks in Ham's F-12 medium containing G418antibiotic. After the cells reached confluency, G418 media was replacedwith Ham's F-12 medium containing 33 ng/ml12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM sodium butyrate(NaB) to induce lytic replication of the virus. Twenty-four hourspost-induction, the media was replaced with complete Ham's F-12 mediawithout G418, TPA, or NaB and cells were incubated for 4 days at 37° C.in a 50% CO₂ incubator. The cell supernatant was collected, centrifuged,and filtered using 0.8 μm filter to remove cell debris. The filteredsupernatant was ultra-centrifuged using a Beckman-Coulter type 19 rotorfor 70 min at 10,000 rpm to pellet the virus. EBV-eGFP virus wastitrated in both HEK-293T cells and Raji cells, and stocks were storedat −80° C. for subsequent experiments.

Generation and Purification of Gp350 Virus-Like Particles.

To generate gp350 VLPs, equal amounts (8 μg/plasmid) of the relevantplasmids (pCAGGS-Newcastle disease virus (NDV) matrix (M), andnucleocapsid proteins (NP), and gp350 ectodomain fused to NDV fusion (F)protein cytoplasmic and transmembrane domains) were co-transfected into80% confluent CHO cells seeded in T-175 cm² flasks; supernatant fromtransfected cells containing VLPs was collected and VLPs were purifiedand composition characterized as previously described (47).

Production of Hybridoma Cell Lines.

Seven days prior to immunization, two eight-week-old BALB/c mice werebled for collection of pre-immune serum. The mice were immunized withpurified UV-inactivated EBV three times (Day 0, 21, and 35), and thenboosted every 7 days three times (Day 42, 49, and 56) with VLPsincorporating gp350 on the surface after Day 35. The mice weresacrificed, and their splenocytes were isolated, purified, and used tofuse with P3X63Ag8.653 myeloma cells at a ratio of 3:1 in the presenceof polyethylene glycol (PEG, Sigma). Hybridoma cells were seeded inflat-bottom 96-well plates and selected in specialized hybridoma growthmedia with HAT (Sigma) and 10% FBS as previously described (80).

Indirect ELISA.

Hybridoma cell culture supernatant from wells that had colony-formingcells were tested for antibody production by indirect ELISA. Briefly,immunoplates (Costar 3590; Corning Incorporated) were coated with 50 μlof 0.5 μg/ml recombinant EBV gp350 ectodomain (Immune TechnologyCorporation) diluted in 1× phosphate buffered saline (PBS, pH 7.4) andincubated overnight at 4° C. After washing three times with 1×PBScontaining 0.05% (v/v) Tween 20 (washing buffer), plates were blockedwith 100 μl washing buffer containing 2% (w/v) bovine serum albumin(BSA) (blocking buffer) then incubated for 1 h at room temperature andwashed as above. 100 μl of hybridoma supernatant or 50 μl of 10 μg/mlpurified mAbs was added to each well (in triplicate) and incubated for 2h at room temperature. Anti-KSHV gH/gL 54A1 and m72A1 mAbs were added asnegative and positive controls, respectively. The plates were washed asdescribed above, followed by incubation with 50 μl of goat anti-mouseIgG HRP-conjugated secondary antibody (1:2,000 diluted in 1×PBS) at roomtemperature for 1 h. The plates were washed again and 100 μl ofchromogenic substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS, Life Science Technologies) was added. The reaction wasstopped using 100 μl of ABTS peroxidase stop solution containing 5%sodium dodecyl sulfate (SDS) in water. The absorbance was read at anoptical density of 405 nm using an ELISA reader (Molecular Devices). Allexperiments were performed in triplicate and confirmed in threeindependent experiments.

Antibody Purification, Quantification, and Isotyping.

Hybridoma cells from selected individual positive clones were expandedstepwise from 96-well plates to T-75 flasks. At confluence in T-75flasks, supernatant from individual clones was collected, clarified bycentrifugation (4,000 g, 10 min, 4° C.), and filtered through a 0.22-μmmembrane filter (Millipore). Antibodies were further purified byCapturem™ Protein A Maxiprep (Takara) and stored in 1×PBS (pH 7.4) at 4°C. Alternatively, antibodies were purified using protein G affinitychromatography followed by size-exclusion chromatography at the BeckmanInstitute of City of Hope X-ray Crystallography Core facility.Antibodies were analyzed by SDS-PAGE to determine purity. Bicinchoninicacid assay (BCA assay; Thermo Fisher Scientific) was conducted todetermine the concentration of purified antibodies. Isotypeidentification was performed with the Rapid ELISA mouse mAb isotypingkit (Thermo Fisher Scientific). Two independent experiments wereperformed.

RNA Extraction, cDNA Synthesis, and Sequencing and Analysis of theVariable Region of the mAbs.

Total RNA was extracted from 1×10⁶ hybridoma cells using the RNeasy MiniKit (Qiagen). Each hybridoma clone cDNA was synthesized in a totalvolume of 20 μl using Tetro Reverse Transcriptase (200 u), RiboSafeRNase Inhibitor, Oligo(dT)18 primer, dNTP mix (10 mM each nucleotide),and 100-200 ng RNA. Reverse transcription was performed at 45° C. for 30min, and terminated at 85° C. for 5 min. The cDNA was stored at −20° C.Immunoglobulin (Ig) VH and VL were amplified using the mouse Ig-specificprimer set purchased from Novagen (48). The VH and VL genes wereamplified in separate reactions and PCR products were visualized on 1%agarose gel.

The VH and VL amplicons were sequenced using an Illumina MiSeq platform:duplicate 50 μl PCR reactions were performed, each containing 50 ng ofpurified cDNA, 0.2 mM dNTPs, 1.5 mM MgCl₂, 1.25 U Platinum Taq DNApolymerase, 2.5 μl of 10×PCR buffer, and 0.5 pM of each primer designedto amplify the VH and VL. The amplicons were purified using an AxyPrepMag PCR Clean-up kit (Thermo Fisher Scientific). The Illumina primer PCRPE1.0 and index primers were used to allow multiplexing of samples. Thelibrary was quantified using ViiA™ 7 Real-Time PCR System (LifeTechnologies) and visualized for size validation on an Agilent 2100Bioanalyzer (Agilent Technologies) using a high-sensitivity cDNA assay.The sequencing library pool was diluted to 4 nM and run on a MiSeqdesktop sequencer (Illumina). The 600-cycle MiSeq Reagent Kit (Illumina)was used to run the 6 pM library with 20% PhiX (Illumina), and FASTQfiles were used for data analysis (81). The determination ofimmunoglobulin families was analyzed using the IMGT/V-QUEST tool 210(www.imgtorg/IMGT_vquest/vquest) (82).

Chimeric mAb Construct Generation.

To generate chimeric mAbs, the VH and VL sequences were cloned into thedual-vector system such as pFUSE CHIg/pFUSE CLIg (InvivoGen), whichexpress the constant region of the heavy and light chains of humanimmunoglobulins, respectively (Genewiz). The constructs were transientlytransfected into HEK-293 6E cells. The supernatants were collected at 72h post-transfection and IgG was purified using protein A/G affinitychromatography.

Humanization of 72A1.

To generate humanized mAbs, the BioLuminate interface (Schrödinger) wasused to identify the human VH and VL framework using 72A1. The resultingmodel was visually inspected to ensure appropriate packing at the baseof the CDR. The sequence was meditope-enabled to add functionality forgenerating bispecific antibodies in the future (83). The resultingsequences were codon-optimized, synthesized, and cloned into the PD2610vector (Atum). The constructs were transiently transfected into ExpiCHOcells following the manufacturer's protocol. Supernatant was collectedat 10 days post-transfection and IgG was purified using protein Gaffinity chromatography, followed by size-exclusion chromatography.

Immunoblot Analysis.

CHO cells were cultured and stably co-transfected with full-lengthpCAGGS-gp350/220 and pCI-puro vector containing a puromycin resistancegene. Forty-eight hours post-transfection, DMEM media containing 10μg/ml of puromycin was added to enrich for cells expressing gp350protein. Puromycin-resistant clones were expanded, followed by flowcytometry sorting using m72A1 to a purity >90%. EBV gp350-positive CHOcells were harvested and lysed in radioimmunoprecipitation assay buffer(RIPA) followed by centrifugation at 15,000 g for 15 min on a benchtopcentrifuge. The lysate was collected and heated at 95° C. for 10 min inSDS sample buffer containing β-mercaptoethanol, then separated usingSDS-PAGE. Proteins were transferred onto a nitrocellulose membrane usingan iBlot™ Transfer System (Thermo Fisher Scientific) followed byincubation in blocking buffer (1% BSA; 20 mM Tris-HCl, pH 7.5; 137 mMNaCl; and 0.1% Tween-20 [TBST]) for 1 hour. The blots were incubated inTBST containing purified anti-gp350 antibodies (1:50) overnight at 4° C.After three washes with TBST, the blots were incubated withHRP-conjugated goat anti-mouse (1:2000) in TBST for 1 hour. After threewashes, the antibody-protein complexes were detected using the AmershamECL Prime Western Blotting Detection Reagent (GE Healthcare). Allexperiments were independently repeated three times.

Flow Cytometry.

To assess the ability of purified anti-gp350 mAb to detect surfaceexpression of EBV gp350 protein by flow cytometry, CHO cells that stablyexpress EBV gp350 were harvested and stained with purified anti-gp350(10 μg/ml), followed by AF488 goat anti-mouse IgG secondary antibody.Flow cytometric analysis was performed on a C-6 FC (BD Biosciences) anddata was analyzed using FlowJo Cytometry Analysis software (FlowJo, LLC)as described (47). All the experiments were independently repeated threetimes.

EBV Neutralization Assay.

EBV neutralization assay was performed in Raji cells as previouslydescribed (47). Briefly, purified individual anti-gp350 mAbs wereincubated with purified AGS-EBV-eGFP (titer calculated to infect atleast 8% of HEK293 cells seeded in 100 μl of serum-free DMEM) for 2hours at 37° C. To represent EBV infection of B cells, the pre-incubatedanti-gp350 mAbs/AGS-EBV-eGFP were used to infect 5×10⁵ Raji cells seededin a 96-well plate for 2 hours at 37° C. Neutralizing 72A1 andnon-neutralizing 2L10 anti-gp350 mAbs served as positive and negativecontrols, respectively. Infected cells were washed three times with PBSto remove any unbound viruses and antibodies. Washed, infected cellswere incubated in 96-well plates at 37° C. for 48 hours post-infectionand the number of eGFP+ (infected) cells was determined using flowcytometry. All dilutions were performed in quintuplicate and the assayswere repeated three times. Antibody EBV neutralization activity wascalculated as: % neutralization=(EBValone−EBVmAb)/(EBValone)×100.

Epitope Mapping by Competitive Cell Binding Assay.

To evaluate conformation epitope mapping of the selected mAbs,competitive binding assays were conducted using biotinylated mAbs. Aone-step antibody biotinylation kit (MACS Miltenyi Biotec) was used tobiotinylate the mAbs. Approximately 1×10⁵ CHO cells that stablyexpressed EBV gp350 were incubated for 2 hours with serially diluted(500, 250, 125, and 67.5 μg/ml) unlabeled competitor mAbs andnon-specific anti-KSHV gH/gL 54A1 mAb. After being washed with PBS, thecells were incubated for 2 hours in the presence of 1 μg/ml biotinylatedmAbs. To determine maximum binding, cells in which the biotinylated mAbwas added in the absence of unlabeled mAbs were included in the assay.Cells were washed with PBS, followed by incubation for 1 hour withanti-biotin AF488 at a dilution of 1:500. After the final wash in PBS,cells were resuspended in 1% paraformaldehyde and analyzed by flowcytometry as described above. Percentage of inhibition was calculatedas: 100−[(% fluorescent cells with competitor mAb/% fluorescent cellswithout competitor mAb)×100]. The complete prevention of binding of abiotinylated mAb by its unlabeled counterpart was used as a validationof the assay, as previously described (84).

Synthesis of 20-Mer Linear Peptides of Gp350 Proteins.

Forty-five sequential 20-mer linear peptides, covering the wholesequence of gp350 (GenBank: NC_007605.1), with an exception of aa862-881, were synthesized using a solid phase method and cleaved using alow-high hydrogen fluoride method by the GenicBio, as previouslydescribed (58). Synthesis of aa 862-881 (pep-44) was not possible due tomultiple hydrophobic aa.

Linear Epitope Mapping by Peptide-mAb Binding Assay.

The binding of anti-gp350 mAbs to 45 synthesized 20-mer sequentialpeptides covering the total length of gp350 was analyzed using indirectELISA as described (58). Briefly, immunoplates were coated with 50 μl of10 μg/ml EBV gp350 peptides (forty-five 20-mers) diluted in PBS andincubated overnight at 4° C.; 0.5 μg/ml recombinant EBV gp350 ectodomainprotein was used as a positive control. After washing three times withwashing buffer (PBS containing 0.05% (v/v) Tween 20), plates wereblocked with 100 μl washing buffer containing 3% BSA (blocking buffer),incubated for 1 hour at room temperature, and washed as above. Ten pg/mlpurified mAbs were added to each well in triplicate and incubated for 2hours at room temperature. Anti-gp350 antibodies m72A1 and h72A1 wereadded as positive controls and anti-KSHV-gH/gL 54A1 mAb was used asnegative control. The plates were washed as described above, followed byincubation with goat anti-mouse IgG or goat anti-human IgGHRP-conjugated secondary antibody (1:2,000 diluted in PBS) at roomtemperature for 1 hour. The plates were washed again and the chromogenicsubstrate ABTS was added. The reaction was stopped using ABTS peroxidasestop solution. The absorbance was read at an optical density of 405 nmusing an ELISA plate reader.

Statistical Analysis.

Unpaired Mann-Whitney U test was used to assess statistical differencesbetween two independent groups. Statistical calculations were performedin Graphpad Prism. Data was considered statistically significant atp<0.05.

Example 2: Novel Anti-Gp350 mAbs Target Linear and ConformationalEpitopes

New EBV gp350-specific mAbs were generated and biochemicallycharacterized, and their ability to neutralize EBV infection wasevaluated. In addition, the antibodies were used to map immunodominantepitopes on the EBV gp350 protein. 23 novel monoclonal antibodiesspecific against EBV gp350 were developed. To generate hybridomas,BALB/c mice were immunized three times with purified UV-inactivated EBVand boosted three times with virus-like particles (VLPs) thatincorporate EBV gp350 ectodomain (1-841) on the surface to improveantibody affinity maturation and avidity. Then the splenocytes wereisolated from the immunized mice and fused with myeloma cells togenerate hybridomas. Specifically, five eight-week-old BALB/c mice wereimmunized with virus-like particles incorporating gp350/220 on thesurface, four times (day 0, 14, 28, and 56) via intraperitonealinjection without adjuvants. At day 64, immunized mice were boosted onceintravenously. Animals were sacrificed at Day 70 to harvest splenocytesfor fusion with the mouse myeloma P3X63Ag8 cell line.

Indirect ELISA was used to screen supernatants from the hybridomas forspecificity against purified EBV gp350 ectodomain protein (AA 4-863) and23 hybridomas producing gp350 specific antibodies were identified. Tofurther characterize the biochemical properties of the 23 antibodiesgenerated, the antibodies were purified from the hybridoma supernatantsusing protein A spin columns, followed by SDS-PAGE to confirm the purityof all antibodies (FIG. 1A).

These novel antibodies were analyzed by flow cytometric analysis forsurface expression of gp350 protein on 10⁶ CHO cells transfected with 1μg of pCAGGS-gp350. gp350 expressing cells were resuspended in PBS,stained with anti-gp350 mAb, which detects the gp350 ED, followed bysecondary Ab goat anti-mouse conjugated to AF488. Additionally, westernblot analysis was conducted on untransfected and pCAGGS-gp350transfected CHO lysate. Anti-gp350 mAb 72A1 was used as a positivecontrol (1:100) and anti-gp350 hybridoma clone supernatants were used at1:50, and anti-mouse secondary antibody was used at 1:2000.

It was found that all 23 hybridoma producing antibodies, designatedHB1-23, recognized the gp350 antigen in an initial ELISA screening usingunfractionated and unpurified hybridoma supernatants (data not shown).When the quantified amount of the purified antibodies (10 μg/ml) wasreevaluated using indirect ELISA, all of the 23 antibodies and m72A1(anti-gp350 positive control) had ELISA signals greater than two timesthose of anti-KSHV gH/gL mAb 54A1 (negative control), and wereconsidered positive/specific to gp350 (FIG. 1B). Of the 23gp350-positive hybridoma producing antibodies identified, HB4, HB5, HB7,HB13, and HB14 demonstrated binding strength equal to or greater thanthat of the positive control, m72A1. This difference in binding of theantibodies could be due to differential exposure of cognate epitopes ongp350 in the assay performed.

Determining the nature of the binding between an antibody and its targetantigen is an important consideration for the performance andspecificity of an antibody, as it can involve the recognition of alinear or conformational epitope (49). The ability of the purifiedantibodies to bind linear epitopes was evaluated by performingimmunoblot analysis of denatured gp350 antigen expressed on Chinesehamster ovary (CHO) cells, and 16 of the 23 antibodies detected both the350 kDa and the 220 kDa splice variant. In contrast, HB2, HB3, HB6, HB7,HB13, HB20, and HB21, as well as the negative control 54A1, failed torecognize either of the denatured isoforms of gp350 (FIG. 1C). Theantibodies' ability to bind conformational epitopes was furthercharacterized by flow cytometric analysis of CHO cells stably expressinggp350 on the cell surface. HB1, HB2, HB3, HB5, HB6, HB9, HB11, HB12,HB15, HB17, HB19, HB20, HB21, and m72A1 antibodies readily recognizedgp350 (FIGS. 1D and 1E). The fact that HB2, HB3, HB20, and HB21 detectedgp350 by flow cytometry, but not by immunoblot, suggests that these fourantibodies only recognized conformational epitopes (i.e., native) ongp350, whereas HB5, HB9, HB11, HB15, HB17, and HB19 recognized bothlinear and conformational epitopes (FIGS. 1C-1E). The observation thatall 23 anti-gp350 antibodies recognized the gp350 antigen either byindirect ELISA, flow cytometry, or immunoblot assay suggests thatantibodies that are specific to EBV gp350 protein were successfullyproduced. In addition, the isotypes of the newly generated antibodieswere determined to be IgG1 (n=14), IgG2a (n=5), IgG2b (n=1), a mixtureof IgG1 and IgG2b (n=1), and a mixture of IgG1 and IgM (n=2) (Table 4).

TABLE 4 Summarized biochemical and functional characterization ofanti-gp350 antibodies ELISA binding Flow Anti- IgG Light to purified EBVcytometry Western body sub-class chain gp350/220 (CHO Cells) blot HB1IgG1 κ + + + HB2 IgG2a κ + + − HB3 IgG2a κ + + − HB4 IgG1 κ + − + HB5IgG2a κ + + + HB6 IgG1 κ + + − HB7 IgG1 κ + − − HB8 IgG1 κ + − + HB9IgG2a κ + + + HB10 IgG1 κ + − + HB11 IgG1 κ + + + HB12 IgG1 κ + + + HB13IgG1 κ + − − HB14 IgG1 κ + − + HB15 IgG1 κ + + + HB16 IgG1/IgM κ + − +HB17 IgG2b κ + + + HB19 IgG1/IgM κ + + + HB20 IgG2a κ + + − HB21IgG1/IgG2b κ + + − HB22 IgG1 κ + − + HB23 IgG1 κ + + + m72A1 IgG1κ/λ + + +

Example 3: Identification and Characterization of 15 Novel Anti-350Monoclonal Antibodies

To determine whether the generated hybridomas were monoclonal or amixture of antibodies, the VH and VL variable region genes of the 23 newanti-gp350 antibodies, as well as m72A1 (positive control), weresequenced using Illumina MiSeq. The sequence of the CDR of m72A1antibody was recently determined and published (50, 51). PCR was used toamplify the genes encoding the VH and VL chain regions in cDNAsgenerated from the 23 hybridoma cells, as well as from m72A1. The PCRproducts presented distinct bands for VH (˜350-450 bp) and VL (˜450-500bp). FIG. 2 is a representation of identified bands for only a fewselected hybridomas which did not yield additional multiple non-specificbands from PCR amplifications that required extra-purification steps.The purified fragments were sequenced, followed by in silico analysis,and CDRs for both VH and VL were identified (Table 1). As previouslyreported, two unique IgG1 VH and two unique VL chains, one kappa and onelambda sequence of m72A1, were identified using the light chain kappadegenerative primers and specific primers for the lambda light chain(50). These sequences were >94% identical to the previously publishedsequences, suggesting that m72A1 exists as a mixture of antibodies,instead of the reported mAb (51). Similar to m72A1, HB4, HB13, HB15, andHB23 hybridomas each produced a mixture of two antibodies, with twounique sequences of the VH chain showing at >5% frequencies, suggestingthat they are not mAbs (Table 5). Coding sequences for VL chains forHB7, HB9, and HB17 were unable to be identified, unless the frequencieswere lowered to >1% (Table 5); in this case, the identified coding VLchain sequences were identical.

TABLE 5 Summary of Illumina Dual Demultiplex of V_(H) and V_(L)Regions >5% PEAR Length Primer >5% Unique Starting Merged FilteredMatched 3× Unique Non- Sample Chain Pairs Reads Reads Reads Reads CodingCoding HB1 HEAVY 51,641 51,210 46,655 32,482 22,725 1 0 LIGHT 280,048279,012 100,725 68,041 58,570 1 1 HB2 HEAVY 22,793 22,621 16,475 11,4157,429 1 0 LIGHT 167,230 166,496 161,764 132,752 115,886 1 1 HB3 HEAVY26,382 26,162 25,542 16,910 11,709 1 0 LIGHT 12,681 12,609 11,753 9,8098,023 1 1 HB4 HEAVY 38,811 38,238 17,151 11,957 7,217 2 0 § LIGHT179,249 129,752 111,996 78,392 66,419 1 1 HB.5 HEAVY 42,951 42,17335,793 25,842 17,173 1 0 LIGHT 176,073 175,267 168,806 141,712 127,045 11 HB6 HEAVY 26,142 25,981 22,245 15,658 10,453 1 0 LIGHT 171,996 171,370167,730 138,348 122,397 1 1 HB7 HEAVY 32,443 32,094 25,449 17,615 11,8361 0 LIGHT 67,031 63,924 37,344 26,271 22,378 1 1 * HB8 HEAVY 140,091103,349 92,744 58,292 31,583 1 0 LIGHT 151,244 115,527 102,439 82,15470,803 1 1 HB9 HEAVY 37,057 36,473 19,544 11,585 7,358 1 0 LIGHT 409,432310,529 136,074 106,820 90,063 1 2 * HB10 HEAVY 38,181 37,981 26,04317,104 11,391 1 0 LIGHT 114,255 112,498 106,914 84,368 75,370 1 1 HB11HEAVY 22,225 21,841 6,956 4,408 2,465 1 0 LIGHT 106,465 102,278 65,33250,232 44,527 1 0 HB12 HEAVY 83,044 82,355 46,350 30,276 20,886 1 0LIGHT 53,098 47,336 15,823 7,560 5,845 1 1 HB13 HEAVY 81,451 80,37247,995 32,216 20,139 2 0 § LIGHT 27,314 24,774 8,987 5,457 4,104 2 1HB14 HEAVY 76,299 75,357 28,309 19,104 12,939 1 0 LIGHT 153,011 149,26448,474 29,710 25,133 1 1 HB.15 HEAVY 26,551 26,410 16,387 11,434 7,002 20 § LIGHT 78,525 77,778 43,509 29,504 24,731 1 1 HB16 HEAVY 54,24953,943 9,517 7,128 4,179 1 0 LIGHT 42,048 40,351 30,602 22,758 18,251 21 § HB17 HEAVY 111,614 110,882 81,428 50,844 35,949 1 0 LIGHT 102,490100,488 83,925 65,925 57,727 1 1 * HB18 HEAVY 211,215 155,410 146,25691,009 50,308 1 0 LIGHT 212,261 161,879 155,235 123,096 105,959 1 1 HB19HEAVY 109,692 82,221 20,546 12,587 7,274 1 1 LIGHT 70,828 69,744 62,57248,354 42,051 1 1 HB20 HEAVY 15,781 15,632 12,789 7,757 4,852 1 0 LIGHT135,527 133,208 118,513 90,717 78,701 1 1 HB21 HEAVY 15,312 15,202 8,5775,645 3,420 1 0 LIGHT 102,450 100,171 89,059 68,552 60,500 1 1 HB22HEAVY 217,959 156,488 154,008 95,755 50,245 1 0 LIGHT 205,334 156,986143,386 108,728 85,136 1 0 HB23 HEAVY 196,390 143,929 123,028 71,07639,358 2 0 § LIGHT 158,594 120,140 115,476 90,004 78,787 2 0 72A1 HEAVY213,480 158,199 156,215 107,395 68,486 2 0 § LIGHT 187,216 140,964132,945 105,783 91,208 1 1 * Hybridoma with V_(L) chain sequencesidentified with >1% frequency, § Hybridoma with more than one unique,plausible-coding V_(H) chain sequence with >5% frequency. The term“unique” refers to unique sequence counts (so, identical sequences foundin a substantial frequency of merged reads, not necessarily uniquecompared to other samples).

The analysis and comparison of the VH and VL chain gene sequences of the23 hybridomas compared to m72A1 showed unique sequences within the CDR1-3 region (Table 1). Only HB8 and HB18 had identical VH and VL chaingene sequences, suggesting that the two are the same clone isolatedseparately; therefore, HB18 was excluded from subsequent experiments.One of the two paired sequences from HB15 hybridoma mixture wasconfirmed to have identical VH and VL gene sequences to that of mAbHB10; however, based on the previous characterization, the presence ofthe additional paired sequenced in the HB15 hybridoma was sufficient toconfer subtle differences in biochemical interactions with gp350 betweenthe HB10 and HB15 purified antibodies. In addition, the germline genesfor the VH and VL chains of the new 15 anti-gp350 mAbs and m72A1 weredetermined (Table 6).

TABLE 6 Identification of the germline genes for VH and VL of 15 newmAbs and m72A1 Anti- V-GENE J-GENE V-GENE J-GENE bodies and allele andallele and allele and allele HB1 IGHV2-9*02 IGHJ4*01 IGKV6-15*01IGKJ2*01 HB2 IGHV9-2-1*01 IGHJ4*01 IGKV4-72*01 IGKJ5*01 HB3 IGHV1S81*02IGHJ4*01 IGKV10-96*01 IGKJ1*01 or IGKJ1*02 HB5 IGHV1S45*01 IGHJ3*01IGKV5-48*01 IGKJ5*01 HB6 IGHV9-2-1*01 IGHJ4*01 IGKV4-72*01 IGKJ5*01 HB7IGHV1-15*01 or IGHJ1*01 IGKV6-13*01 IGKJ5*01 IGHV1-23*01 HB8IGHV9-3-1*01 IGHJ3*01 IGKV1-117*01 IGKJ2*01 HB9 IGHV5-6-4*01 IGHJ1*01IGKV1-117*01 IGKJ2*01 HB10 IGHV1S81*02 IGHJ4*01 IGKV10-96*01 IGKJ1*01 orIGKJ1*02 HB11 IGHV3-8*02 IGHJ1*01 IGKV4-50*01 IGKJ1*01 HB12 IGHV1S81*02IGHJ4*01 IGKV1-110*01 F IGKJ4*01 HB14 IGHV1-15*01 IGHJ1*01 GKV1-110*01 FIGKJ4*01 HB17 IGHV1S81*02 IGHJ4*01 IGKV10-96*01 IGKJ1*01 or IGKJ1*02HB20 IGHV1S81*02 IGHJ4*01 IGKV10-96*01 IGKJ1*01 or IGKJ1*02 HB22IGHV2-6*02 IGHJ3*01 IGKV1-117*01 IGKJ1*01 m72A1 IGHV1-18*01 or IGHJ3*01IGLV1*01 IGLJ1*01 IGHV1-26*01

These results show that although only two mice were used in thegeneration of the antibodies, germline diversity was still present tosome extent, and few mAbs shared the same germline gene rearrangementand evolution. Thus, based on the sequence analysis (FIG. 3 ), 15 uniqueanti-gp350 mAbs were generated, with distinct sequence identities fromcommercially available m72A1. The sequence of the widely usednon-neutralizing antibody 2L10 (originally from G. Pearson's laboratory)was not available and therefore, 2L10 was not used in the sequencecomparative studies.

Example 4: Humanization of m72A1 and HAMA Testing

The m72A1 VH and VL chain sequences identified in this study wereidentical to the ones published by Herrman et al., and they were used togenerate a humanized 72A1 352 (h72A1) as a strategy to reduce and/oreliminate HAMA (FIG. 4A). The disclosed h72A1 bound gp350 with similarstrength to m72A1 in both ELISA (FIG. 4B). The levels of anti-mouse andanti-human activity retained in the h72A1 nAb were determined usingELISA. As shown in FIG. 4C, mouse 72A1 mAb reacted strongly togoat-anti-mouse IgG as compared to goat anti-human IgG (9-fold,p<0.0001) and 28-fold above the background (1×PBS) (p<0.0001). Incontrast, h72A1 mAb did not react at all to goat anti-mouse, butspecifically reacted strongly to goat anti-human IgG (2100-fold,p<0.0001) over the background. To determine whether h72A1 stillrecognized gp350 in its native conformation, flow cytometric analysiswas performed. h72A1 recognized native epitopes of gp350 expressed onCHO cells surface, comparable to m72A1 360 (FIG. 4D). These resultsindicate that humanization of m72A1 did not affect its ability torecognize native gp350, but it abrogated anti-mouse reactivity andincreased anti-human reactivity.

Example 5: Neutralization Assay

Currently, m72A1 is the only commercially available anti-gp350 nAb (68).However, this antibody was recently reported to be a mixture of bothfunctional and non-functional antibodies (50). The ability of the 15 newmAbs (10 μg/ml or 50 μg/ml) to neutralize purified eGFP-tagged-EBVinfection of a B cell line (Raji) in vitro was evaluated and compared tothat of m72A1 (mixture) and the newly cloned and biochemicallycharacterized h72A1, following standardized procedures (47, 52). Thepercentage of eGFP+ cells (percent infection) was determined using flowcytometry as described (47). The nAbs 72A1 and E1D1 were used aspositive controls, whereas the anti-gp350 non-neutralizing mAb 2L10 andKSHV gH/gL mAb 54A1 were used as negative controls. Because HB4, HB13,HB15, HB16, HB19, HB21, and HB23 were confirmed to be polyclonal basedon isotyping and/or sequence data, they were eliminated from furtherconsideration in the neutralization assay. HB18 was not used inneutralization experiments because it was identical to HB8.

The purified eGFP-tagged-EBV was titered in Raji cells to determinepercent EBV infection using a range of volumes (50-250 μl) (FIG. 5A).Then initial neutralization of EBV was conducted in Raji cells usingpurified mAbs at various concentrations (12.5, 25, 50, and 100 μg/ml).Only HB1, HB5, HB11, 375 HB22 and m72A1 showed a dose-dependentneutralization of EBV in Raji cells; the neutralization capability ofHB5 (60-80%) was comparable to that of m72A1 (35-80%) (FIG. 5B). Incontrast, HB2, HB3, HB6, HB7, HB8, HB9, HB10, HB12, HB14, HB17, and HB20mAbs failed to neutralize EBV infection, even at the highestconcentration. As expected, neither 2L10 nor 54A1 neutralized EBVinfection, even at the highest mAb concentration of 100 μg/ml (FIG. 5B).

Subsequently, seven representative novel nAb and non-nAb anti-gp350 mAbs(HB1, HB5, HB10, HB11, HB17, HB20 and HB22) were purified as well ascontrols (m72A1, h72A1 and 54A1) using protein G affinity chromatographyand size-exclusion chromatography in order to eliminate any potentialimpurities, then their potency in blocking EBV infection of Raji cellswas reevaluated. Chromatography-purified HB1, HB5, HB11, and HB20,blocked EBV infection in a dose-dependent manner (FIG. 5C). HB5 was themost effective nAb, efficiently blocking EBV infection (90%) atpercentages comparable to both m72A1 (93%) and h72A1 (98%), even at thelowest concentration of 12.5 μg/ml, with 97% nAb activity at 100 μg/ml(FIG. 5C). HB1, HB11, and HB20 neutralized EBV infection between 57-73%at the lowest concentration (12.5 μg/ml) and 90% at 100 μg/ml. NeitherHB17, HB22, nor 54A1 blocked EBV infection; although HB10 blocked someEBV infection, nAb activity did not reach 50% even at the highestconcentration of antibody used, thus it was classified as a non-nAb.

Example 6: Four Novel Gp350 nAbs Bind Antigenic Epitopes that Overlapwith Those of 72A1

At least seven unique CD21 binding epitopes on EBV gp350 have beenpredicted (Table 3). One of these epitopes (AA 142-161) has beenidentified as the primary epitope recognized by m72A1 (59) and miceimmunized with the 142-161 peptide elicit nAbs against EBV infection(51). To evaluate whether the selected novel nAbs (HB1, HB5, HB11, andHB20) and non-nAbs (HB10, HB17, and HB22) bind overlapping ornon-overlapping target epitopes to those of 72A1, their ability tocompete for binding to gp350 expressed stably on transfected CHO cellswere determined. Antigen binding competition was observed betweenbiotinylated m72A1 (1 μg/ml) and serially diluted (500, 250, 125, and67.5 μg/ml) unlabeled gp350 nAbs (HB1, HB5, HB11, HB20, and h72A1), butnot the gp350 non-nAbs (HB10, HB17, or HB22) or anti-KSHV gH/gL antibody54A1 (negative control) (Table 7).

TABLE 7 Cell binding mAbs competition assay with EBV gp350 usingbiotinylated m72A1 % inhibition of biotinylated nAbs Unlabeled 500 250125 67.5 mAbs μg/ml μg/ml μg/ml μg/ml HB1 91 89 81 81 HB5 95 93 92 94HB10 12 14 16 13 HB11 95 95 89 87 HB17 32 32 6 0 HB20 96 91 87 89 HB2210 3 16 9 m72A1 91 93 94 97 h72A1 98 95 93 89 54A1 18 7 10 4

However, previously non-nAbs HB10 and HB22, were shown not to bindnative gp350 expressed on CHO cells using FACS (FIG. 1D), suggestingthat the observed lack of competitive binding could be attributed tothese two Abs not binding the native gp350 expressed on the CHO cells.These results indicate that nAbs HB1, HB5, HB11, and HB20, as well ash72A1, bind overlapping target epitopes with that of m72A1, whilenon-nAbs HB17, the only non-nAbs able to recognize gp350 inconformational form, binds different target epitopes. Similar resultswere obtained when cross-competition binding assays between 1 μg/mlbiotinylated and 500 μg/ml unlabeled gp350 nAbs HB1, HB5, HB11, HB20,m72A1, and h72A1 and non-nAb HB17 were performed (Table 8), confirmingthat the newly developed gp350 nAbs bind overlapping epitopes to 72A1.

TABLE 8 Cross-competitive binding of EBV gp350 % inhibition ofbiotinylated nAbs Unlabeled mAbs HB1 HB5 HB10 HB11 HB17 HB20 HB22 m72A1h72A1 54A1 HB1 92 97 ND 96 16 61 ND 98 95 4 HB5 92 98 ND 93 9 73 ND 9797 13 HB10 ND ND ND ND ND ND ND ND ND ND HB11 93 96 ND 96 17 3 ND 97 979 HB17 23 32 ND 11 86 17 ND 24 16 0 HB20 88 97 ND 89 13 59 ND 98 96 0HB22 ND ND ND ND ND ND ND ND ND ND m72A1 82 97 ND 90 37 31 ND 98 98 1h72A1 87 97 ND 79 31 26 ND 98 98 0 54A1 0 0 ND 0 0 0 ND 0 3 0 ND—Notdetermined

Because of inability of non-nAbs, HB10 and HB22 to recognizeconformational gp350, they were excluded from the cross competitive cellbinding assays. Importantly, even though HB1, HB5, HB11, and HB20competed with 72A1 for the same antigenic epitope, each of these nAbshad unique VH and VL sequences from 72A1 (Table 1, Table 2).

Example 7: Novel Anti-Gp350 nAbs and Non-nAbs Bind Three MajorImmunodominant Regions on Gp350

To identify linear epitopes on gp350, anti-gp350 nAbs (HB1, HB5, HB11and HB20) and non-nAbs (HB10, HB17, and HB22) were scanned in anELISA-based assay using a peptide library consisting of sequentialpeptides (FIGS. 6A-6I). The peptide library, consisting of 20-merpeptides, covered the entire gp350 protein, with the exception of AA862-881 (Table 9).

TABLE 9 Sequence, length and position of EBV gp350 peptides PeptidePeptide gp350 Peptide name position region Peptide sequence length Pep-1 1-20 1 MEAALLVCQYTIQSLIHL 20 TG (SEQ ID NO: 132) Pep-2 21-40 1EDPGFFNVEIPEFPFYPT 20 ON (SEQ ID NO: 133) Pep-3 41-61 1VCTADVNVTINFDVGGKK 21 HQL (SEQ ID NO: 134) Pep-4 62-81 1DLDFGQLTPHTKAVYQPR 20 GA (SEQ ID NO: 135) Pep-5  82-101 1FGGSENATNLFLLELLGA 20 GE (SEQ ID NO: 136) Pep-6 102-121 2LALTMRSKKLPINVTTGE 20 EQ (SEQ ID NO: 137) Pep-7 122-141 2QVSLESVDVYFQDVFGTM 20 WC (SEQ ID NO: 138) Pep-8 142-161 2HHAEMQNPVYLIPETVPY 20 IK (SEQ ID NO: 139) Pep-9 162-181 2WDNCNSTNITAVVRAQGL 20 DV (SEQ ID NO: 140) Pep-10 182-201 2TLPLSLPTSAQDSNFSVK 20 TE (SEQ ID NO: 141) Pep-11 202-221 3MLGNEIDIECIMEDGEIS 20 QV (SEQ ID NO: 142) Pep-12 222-241 3LPGDNKFNITCSGYESHV 20 PS (SEQ ID NO: 143) Pep-13 242-261 3GGILTSTSPVATPIPGTG 20 YA (SEQ ID NO: 144) Pep-14 262-281 3YSLRLTPRPVSRFLGNNS 20 IL (SEQ ID NO: 145) Pep-15 282-301 3YVFYSGNGPKASGGDYCI 20 QS (SEQ ID NO: 146) Pep-16 302-321 4NIVFSDEIPASQDMPTNT 20 TD (SEQ ID NO: 147) Pep-17 322-341 4ITYVGDNATYSVPMVTSE 20 DA (SEQ ID NO: 148) Pep-18 342-361 4NSPNVTVTAFWAWPNNTE 20 TD (SEQ ID NO: 149) Pep-19 362-381 4FKCKWTLTSGTPSGCENI 20 SG (SEQ ID NO: 150) Pep-20 382-401 4AFASNRTFDITVSGLGTA 20 PK (SEQ ID NO: 151) Pep-21 402-421 5TLIITRTATNATTTTHKV 20 IF (SEQ ID NO: 152) Pep-22 422-441 5SKAPESTTTSPTLNTTGF 20 AD (SEQ ID NO: 153) Pep-23 442-461 5PNTTTGLPSSTHVPTNLT 20 AP (SEQ ID NO: 154) Pep-24 462-481 5ASTGPTVSTADVTSPTPA 20 GT (SEQ ID NO: 155) Pep-25 482-501 5TSGASPVTPSPSPWDNGT 20 ES (SEQ ID NO: 156) Pep-26 502-521 6KAPDMTSSTSPVTTPTPN 20 AT (SEQ ID NO: 157) Pep-27 522-541 6SPTPAVTTPTPNATSPTP 20 AV (SEQ ID NO: 158) Pep-28 542-561 6TTPTPNATSPTLGKTSPT 20 SA (SEQ ID NO: 159) Pep-29 562-581 6VTTPTPNATSPTLGKTSP 20 TS (SEQ ID NO: 160) Pep-30 582-601 6AVTTPTPNATSPTLGKTS 20 PT (SEQ ID NO: 161) Pep-31 602-621 7SAVTTPTPNATGPTVGET 20 SP (SEQ ID NO: 162) Pep-32 622-641 7QANATNHTLGGTSPTPVV 20 IS (SEQ ID NO: 163) Pep-33 642-661 7QPKNATSAVTTGQHNITS 20 SS (SEQ ID NO: 164) Pep-34 662-681 7TSSMSLRPSSNPETLSPS 20 TS (SEQ ID NO: 165) Pep-35 682-701 7DNSTSHMPLLTSAHPTGG 20 EN (SEQ ID NO: 166) Pep-36 702-721 8ITQVTPASISTHHVSTSS 20 PA (SEQ ID NO: 167) Pep-37 722-741 8PRPGTTSQASGPGNSSTS 20 TK (SEQ ID NO: 168) Pep-38 742-761 8PGEVNVTKGTPPQNATSP 20 QA (SEQ ID NO: 169) Pep-39 762-781 8PSGQKTAVPTVTSTGGKA 20 NS (SEQ ID NO: 170) Pep-40 782-801 8TTGGKHTTGHGARTSTEP 20 TT (SEQ ID NO: 171) Pep-41 802-821 9DYGGDSTTPRPRYNATTY 20 LP (SEQ ID NO: 172) Pep-42 822-841 9PSTSSKLRPRWTFTSPPV 20 TT (SEQ ID NO: 173) Pep-43 842-861 9AQATVPVPPTSQPRFSNL 20 SM (SEQ ID NO: 174) Pep-44 862-881 9LVLQWASLAVLTLLLLLV 20 MA (SEQ ID NO: 175) Pep-45 882-901 9DCAFRRNLSTSHTYTTPP 20 YD (SEQ ID NO: 176) Pep-46 888-907 9NLSTSHTYTTPPYDDAET 20 YV (SEQ ID NO: 177)

This peptide could not be synthesized due to high hydrophobicity of AAresidues in the sequence. Purified m72A1 and h72A1 nAbs were used aspositive controls and anti-KSHV gH/gL 54A1 antibody as a negativecontrol. Purified recombinant gp350 ectodomain was used as a control tovalidate the binding activity for all of the antibodies used. Theoverall gp350 sequence was divided into nine different regionsconsisting of ˜100 AA (FIG. 7 ). The three major regions that exhibitedthe greatest affinity to anti-gp350 mAbs were: 1-101, 102-201, and402-501 (Table 10).

TABLE 10 Summarized analysis of anti-gp350 mAb linear epitope binding tovarious regions of gp350 gp350 Regions mAbs 1 2 3 4 5 6 7 8 9 HB1 x — —— x x — x — HB5 x x x x x x x — — HB10 x — — — x — x x x HB11 x x — — x— x — — HB17 x — — — — x — — x HB20 x x x — x — — — — HB22 x — — — — — —— — m72A1 x x — — x — — — — h72A1 x x — — x — — — — (X) representspositive binding of antibody to the region, (—) represents no binding ofantibody to the region

The AA 102-201 region was bound by only nAbs (HB5, HB11, HB20, m72A1 andh72A1), with the exception of HB1. Notably, this region (102-201)contains the epitope (AA 142-161) previously identified as a bindingepitope for 72A1 and as a binding receptor for CR2 (Table 3), confirmingthat this is the main region that interacts with most gp350 nAbs.Because both nAbs and non-nAbs bound to AA 1-101 and 402-501, these tworegions were considered to be immunodominant.

Example 8: Construction of Chimeric Gp350 nAbs

Chimeric gp350 nAbs were constructed according to the diagrams of FIG. 8. First, mouse antibodies against human gp350 were developed, and thenthe mouse antibody variable region is fused to human constant region,for example, to human IgG. The heavy chain and light chain variableregions of the mouse antibody were cloned into expression vectors suchas pFUSE-CHIg and pFUSE2-CLIg vectors, respectively, followed byco-transfection of mammalian cells with recombinant pFUSE-CHIg andpFUSE2-CLIg vectors. The expression vectors were obtained fromInvivoGen, and the expression was conducted in CHO cells or HEK293cells, available from ATCC. The construction schemes and expression ofheavy and light chains of clone 19 are shown in FIG. 8 .

Analysis of VH-VL sequence from the HB168 (nAb-72A1) hybridoma revealedthat the hybridoma produced two antibodies: one that is gp350-specificand another that recognizes mineral oil-induced plasmacytoma (MOPC)(57). To further investigate gp350 for additional neutralizing epitopes,the gp350-specific nAb-72A1 VH-VL sequence was used to generate chimeric(mouse/human) recombinant antibodies. Similarly, the VH-VL sequence forthe HB20 antibody, which the neutralization analysis above showed to beone of the best nAb, was used to generate chimeric antibody. A negativecontrol chimeric recombinant antibody was generated using VH-VLsequences from the gp350-specific but non-neutralizing HB5 antibody.

Example 9: Development of Antibody-Small Molecule Conjugates (ADCs)

Using small molecule L₂P₄ as an example, antibody-small moleculeconjugates can be developed as illustrated in FIG. 9 . One or more smallmolecules can be conjugated to the antibody heavy chain or light chainvia a reactive donor group and a C-terminal acceptor group. Smallmolecule L₂P₄ was disclosed in the publication by Jiang et al.⁴⁰ Aftervalidating the function of the purified chimeric gp350 nAbs using ELISA,flow cytometry (FC), and surface plasmon resonance, the nAbs can beconjugated to L₂P₄ in a site-specific manner via a Val-Cit dipeptidelinker, which releases the active agent upon internalization of theADC.^(41, 42)

Example 10: Efficacy Test of ADCs

To screen for optimal dose and identify the best nAb clone forpre-clinical studies, the ability of the ADC to neutralize EBV in vitroand to protect against PTLD in vivo is tested using a humanized mouse,as described^(43,44). In brief, purified chimeric ADC (or controls: PBS,or isotype-matched non-nAbs) is injected by I.V. into humanized mice,followed by EBV-B95-8-eGFP challenge. Mice are monitored regularly andeuthanized upon signs of illness or after a preset limit of 100 days.Routine histology and necropsy are conducted to assess the efficacy ofthe ADC to protect against EBV infection and PTLD development.

Example 11: Humanization of E1D1

Similar to Example 4, humanized E1D1 antibody was produced and itsspecificity was compared to mouse E1D1 by flow cytometric analysis (FIG.10 ). Humanization of E1D1 did not affect its binding specificity to EBVgH/gL.

Example 12: Biochemical Characterization of Chimeric and HumanizedAntibodies

As demonstrated by FIG. 11 , murine, chimeric and humanized antibodiespurified by affinity and size-exclusion chromatography were analyzed bySDS-PAGE under reducing conditions. Protein band sizes of 50 kDa and 25kDa corresponding to the heavy and light chain, respectively wereobserved. ELISA was performed to determine the reactivity of chimericand humanized antibodies to murine IgG. Soluble EBV gp350 or gH/gLprotein was used as the target antigen at 0.5 μg/ml. Plates wereincubated with 0.5 μg/ml of primary antibody, followed by three washes.Bound antibodies were detected using HRP-conjugated anti-mouse IgG oranti-human IgG (1:2,000). The chimeric and humanized antibodies did notreact to murine IgG. To determine whether chimeric and humanizedanti-gp350 retained the ability to recognize linear epitopes on gp350,lysate from CHO WT cells and CHO cell stably expressing gp350 wereanalyzed by western blot analysis. The chimeric and humanization processof anti-gp350 did not affect the binding affinity of the antibodies togp350 linear epitopes, as indicated by the protein band observed at ˜350kDa.

ELISA binding of anti-gp350 and anti-gH/gL to soluble gp350 and gH/gLproteins was performed. Soluble EBV gp350 and gH/gL proteins were usedas the target antigen at 0.5 μg/ml. 1× phosphate buffered saline (PBS)was used as negative (not shown) control. Bound antibodies were detectedusing HRP-conjugated anti-mouse or anti-human IgG (1:2,000). Thechimeric and humanization process did not affect the ability ofanti-gp350 and anti-gH/gL to bind to purified recombinant gp350 andgH/gL proteins, respectively.

Flow cytometric analysis of gp350 and gH/gL specificity was performed.CHO wild type cells and gp350 or gH/gL-expressing CHO cells were stainedwith primary antibodies, followed by secondary goat anti-mouse oranti-human conjugated to AF488. Unstained cells and cells stained withsecondary goat anti-mouse or anti-human conjugated to AF488 alone, wereused as negative controls. Binding of chimeric and humanized anti-gp350or gH/gL to conformational epitopes was comparable to that of theparental murine antibodies.

Example 13: EBV Inhibitory Effects of Anti-Gp350 and Anti-gH/gL nAbs

As shown in FIG. 12 , EBV-eGFP was pre-incubated with serial diluted(10-0.01 μg/ml) purified murine, chimeric and humanized anti-gp350 (HB5and 72A1) and anti-gH/gL (E1D1) followed by incubation for 24 hours with2.5×10⁵ HEK 293 (12A) or SVKCR2 cell (12B) or 5×10⁵ Raji cells (12C).EBV-eGFP+ cells were enumerated using flow cytometry. Murine nAb (mHB5,m72A1 and mE1D1) served as positive controls. h72A1 and chHB5 were ableto neutralize EBV infection in both B cells (Raji cells) and CR2+epithelial cells (HEK 293 and SVKCR2 cells) similar to or better thanthe murine versions, m72A1 and mHB5.

REFERENCES

The references listed below, and all references cited in thespecification are hereby incorporated by reference in their entireties,as if fully set forth herein.

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1. An antibody or an immunogenic fragment thereof that specificallybinds to Epstein-Barr virus (EBV) glycoprotein (gp) 350 or glycoprotein220.
 2. The antibody or the immunogenic fragment thereof of claim 1,wherein the antibody or the immunogenic fragment thereof specificallybinds to a fragment of gp350 comprising residues 1-101, 102-201, or402-501 of SEQ ID NO:
 182. 3. The antibody or the immunogenic fragmentthereof of claim 2, comprising a heavy chain comprising a sequence atleast 95% identical to SEQ ID NOs: 179 or
 184. 4. The antibody or theimmunogenic fragment thereof of claim 3, comprising a light chaincomprising a sequence at least 95% identical to SEQ ID NOs: 181 or 186.5. The antibody of claim 4, wherein the antibody is a monoclonalantibody.
 6. The antibody of claim 4, wherein the antibody is a chimericantibody, a humanized antibody or a human antibody.
 7. The antibody ofclaim 6, wherein the antibody is humanized 72A1 or humanized E1D1.
 8. Animmunogenic peptide comprising one or more fragments of gp350, whereineach fragment has an amino acid sequence identical to or sharing atleast 60% similarity to residues 1-101, 102-201, or 402-501 of SEQ IDNO:
 182. 9. The immunogenic peptide of claim 8, further comprising aknown immunogenic peptide such as keyhole limpet hemocyanin (KLH)peptide.
 10. An antibody-small molecule conjugate comprising: anantibody or an immunogenic fragment thereof that specifically binds to afragment of EBV gp350 or EBV gp220 comprising residues 1-101, 102-201,or 402-501 of SEQ ID NO: 182; and a small molecule having ananti-proliferative activity against EBV-transformed cells, wherein thesmall molecule is conjugated to the antibody.
 11. The conjugate of claim10, wherein the antibody is a monoclonal antibody.
 12. The conjugate ofclaim 11, wherein the antibody is a chimeric antibody, a humanizedantibody or a human antibody. 13-16. (canceled)
 17. A pharmaceuticalcomposition comprising the antibody or the immunogenic fragment thereofof claim
 4. 18. A method of neutralizing EBV infection comprisingadministering to a subject infected with EBV a therapeutically effectiveamount of the antibody or the immunogenic fragment thereof of claim 4.19. A method of preventing EBV infection comprising administering to asubject at an elevated risk of EBV infection a therapeutically effectiveamount of the antibody or the immunogenic fragment thereof of claim 4.20-24. (canceled)
 25. A method of immunizing or vaccinating a subjectagainst EBV infection comprising administering to the subject atherapeutically effective amount of the antibody or the immunogenicfragment thereof of claim
 4. 26-29. (canceled)
 30. A pharmaceuticalcomposition comprising the immunogenic peptide of claim
 8. 31. A methodof neutralizing EBV infection comprising administering to a subjectinfected with EBV a therapeutically effective amount of the immunogenicpeptide of claim
 8. 32. A method of preventing EBV infection comprisingadministering to a subject at an elevated risk of EBV infection atherapeutically effective amount of the immunogenic peptide of claim 8.33. A method of immunizing or vaccinating a subject against EBVinfection comprising administering to the subject a therapeuticallyeffective amount of the immunogenic peptide of claim 8.